Protein Blotting: A Manual

METHODS OF BIOCHEMICAL ANALYSIS VOLUME 33

Protein Blotting: A Manual

JONATHAN M. GERSHONI, Department of Biophysics, The Weizmunn Institute of Science, Rehovot, Israel

I. 11.

111. IV.

V.

v1.

VII.

VIII. IX. X.

XI.

XII.

Scope Historical Perspective A Typical Blot Experiment To Blot or Not to Blot . . . That Is the Question 1. Quantitative Dot Blotting 2. Gels 1. Sample Preparation 2. Acrylamide Concentration 3. Transfer of Fixed Gels 4. Isoelectrofocusing and Two-Dimensional Gels The Immobilizing Matrix 1. Chemical Composition 2. Physical Characteristics Protein Transfer 1. Diffusion and Convection Blotting 2. Electroblotting

Dot Blotting as a Screening Assay

A. Apparatus Design a. Electrode Design b. The Cassette

B. Transfer Buffers C. The Power Source

Staining of Blots Quenching Probing and Washing Signal Detection 1. Autoradiography 2. Detection of Enzyme Conjugates Correlating a Signal to a Band 1. Mixed Dye Fronts 2. Internal Markers

1

Methods of Biochemical Analysis, Volume 33 Edited by David Glick

Copyright © 1988 by John Wiley & Sons, Inc.

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XIII.

XIV.

xv .

XVI.

JONATHAN M. CERSHONI

5. Cut Corners 4. Hemoglobin Quench Applications 1. Immunoblotting

A. Corollary Applications of Immunoblotting a. b. Detection of Specific Enzymes c. General Protein Stain

The Analysis of Glycoproteins ,

A. Lectin Overlays B. In Situ Enzyme Modification C. Enzyme Hydrazides

A. Limited Denaturation B. Renaturation

4. Nudeic Acid Overlays 5. Additional Applications Suggested Protocols 1. Dot-Blot Protocol 2. Electroblotting

A. Preparation B. Blotting

Affinity Purification of Monospecific Antibodies

2.

5. Ligand Overlays

3. Storage of Blots 4. Processing Blots 5. Detection of Signal

A. Autoradiography B. Detection of Enzyme-Conjugated Probes C. General Protein Staining of Blots

Troubleshooting 1. Transfer Technique

A. Biased Transfer B. Stubborn Bands C. Baldspots D. Distorted Bands

2. Signal Quality 3. Background Problems

A. Uneven Background B. High Background C. False Positives

Concluding Remarks Acknowledgments References

I. SCOPE

Over the past 10 years, blotting procedures have become an essential element in the biochemical analysis of DNA, RNA, proteins, and lipids.

PROTEIN BLOTTING: A MANUAL 3

As could be expected, numerous articles have been written on various technical aspects of these methods and many more have appeared in which blotting, of one type or another, has been employed as a means to study biological problems.

The subject of protein blotting has already been reviewed (Gershoni and Palade, 1983; Haid and Suissa; 1983; Gooderham, 1984; Symington, 1984; Towbin and Gordon, 1984; Bers and Garfin, 1985; Gershoni, 1985; Beisiegel, 1986) and the main points of this technique are well established. Therefore, this chapter is not intended to be an updated and extensive review of the literature, but rather to provide a practical description of how to blot and analyze proteins. By no means should the protocols given here be regarded as the best possible approach. They have been selected because they are generally simple and reliable. They can almost certainly be improved upon and should be adapted to the specific needs of the system being tested. The concepts described should enable the reader to determine whether a given biological system is amenable to blot analyses. Each section deals with a particular step of blotting, and examples are given to demonstrate the variety of approaches and applications that have been adopted. Obviously, the examples cited are only a representative selection of the many articles published.

11. HISTORICAL PERSPECTIVE

The combined use of sodium dodecylsulfate (SDS) with polyacrylamide gel electrophoresis (PAGE), and discontinuous buffer systems (Laemmli, 1970; Neville, 1971) provides the investigator with the means to evaluate the purity or the complexity of protein mixtures being studied. The resolution of the constituents of the samples analyzed has been increased considerably by the development of two-dimensional gel electrophoresis (O’Farrell, 1975). Nonetheless, identification and characterization of individual peptides require the ability to further probe the electro- phoretogram. Thus, various overlay techniques have evolved (Adair et al., 1978; Burridge, 1978; Glenney and Weber, 1980; Carlin et al., 1981; Snabes et al., 1981; Adair, 1982). Overlay of gels with antibodies or lectins, for example, allows the identification of antigens or glycoproteins, respectively. The manipulation of the gels, however, is often cumbersome and not always sufficiently sensitive. Therefore, it was a marked improvement when blot techniques, originally developed for the analysis of DNA (Southern, 1975), were applied to proteins as well (Erlich et al., 1979; Renart et al., 1979; Towbin et al., 1979; Bittner et

4 JONATHAN M. GERSHONI

al., 1980; Bowen et al., 1980). Rapidly, numerous protocols evolved in which almost every possible element that could be modified, was. Various gel systems have been used ranging from acrylamide to agarose, from SDS denaturing to isoelectrofocusing. Different immobilizing matrices were developed, and changes in the buffer systems have been made. Probing the blots has been accomplished with a diversity of ligands, antibodies, and lectins, as well as with nucleic acids. Even the conditions for washing and blocking the filters have been examined. However, no ideal procedure has emerged and most probably none will. Therefore, only guidelines can be prescribed, to be custom tailored to one’s specific needs.

Curiously, “blottologists” are notorious for their need of jargon and the field has been flooded with lab slang. Many of the terms range from the misleading to the uninformative and a few are simply distasteful. Nonetheless, without going into the origins of the terms that have evolved, there are a few that have become generally accepted. Thus, Southern, Northern, and Western blots refer to the blot analysis of DNA, RNA, and protein, respectively. “Immunoblotting” has become the generic term for the analysis of Western blots with antibodies. Dot blotting is the analysis of macromolecules applied directly to the immobilizing matrix as opposed to transferring them from a gel. Table I presents a collection of blot terms and their meanings.

111. A TYPICAL BLOT EXPERIMENT

Before considering the different parameters that can affect blot analysis, it is useful to outline a typical experiment.

In this example, a crude protein mixture is to be analyzed for the purpose of detecting a particular polypeptide, which is “the antigen” for a given antibody.

The protein mixture is first separated into its constituents, most commonly on a SDS-polyacrylamide gel. After electrophoresis, part of the gel may be stained with Coomassie brilliant blue to serve as a reference, and the remainder is used for blotting. A piece of membrane filter, usually nitrocellulose, is applied to the gel and this assembly is then secured in a cassette, which is placed into a transfer apparatus (ostensibly a Plexiglas tank equipped with two electrode arrays). Elec- trotransfer is performed for a number of hours, and then the gel and filter are removed.

The blotted gel may be stained to determine the efficiency of protein elution, while the blot is quenched in a buffer, containing protein a n d


TABLE I

An Unabridged Glossary of Blot Terms

Block See quench. Blot: n. The product of blotting, that is, the transferred immobilized electrophoretic

pattern, also referred to as “replica.” v. The process of transferring macromolecules from gels to an immobilizing matrix. Thus, DNA blotting, RNA blotting, and protein blotting deal with the transfer and immobilization of DNA, RNA, and protein respectively.

Blotch BLO’lTO: Bovine lactotransfer technique optimizer, that is, nonfat dry milk used as a

Capillary blotting:

An unsuccessful blot (D. Lester, personal experience).

quencher (Johnson et al., 1984). Blotting according to Southern (1975), that is, transferring when

the driving force for elution is the convection (movement of fluid) through the gel and filter due to the capillarity of absorbent paper placed on top of the immobilizing matrix.

Colony blot: matrix (usually nitrocellulose) are probed with DNA or RNA probes or specific antibodies to detect desired recombinant transformed bacteria. In the latter case the bacteria are transformed with recombinant-expression vectors and are grown under inductive conditions (Stanley, 1983).

A technique in which colonies of bacteria grown on an immobilizing

Convection blotting: See capillary blotting. Detergent blot: A technique developed to detect membrane proteins. A gel containing

detergent (e.g., Nonidet P-40) is placed between the acrylamide gel to be blotted and the immobilizing matrix. The hydrophobic integral membrane proteins are trapped in the mid-gel while the more hydrophilic proteins pass through this barrier and are caught on the membrane filter (It0 and Akiyama, 1985).

matrices. Application may be achieved with a micropipette, and the droplets (2-5 ~ 1 ) thus form dots. Vacuum manifolds have been developed and are commercially available to perform dot-blot analyses of larger volumes of samples (Hawkes et al., 1982).

covalently linked to cellulosic paper. The diazo groups provide the ability to covalently immobilize the blotted macromolecules (Alwine et al., 1979).

DPT: 1983); see DBM.

Diffusion blotting: The process of transferring macromolecules from gels to immobilizing matrices by way of diffusion. In such instances two identical blots can be obtained (Bowen et al., 1980).

Malamud, 1982b).

Dot blot: The process of analyzing samples that are directly applied to immobilizing

DBM: Diazobenzyloxymethyl paper, an immobilizing matrix in which diazo groups are

Diazophenylthioether paper (Reiser and Wardale, 1981; Reiser and Stark,

Eastern blotting: Blotting of proteins from isoelectrofocusing gels (Reinhart and

EITB: Enzyme-linked immunoelectrotransfer blot technique (Tsang et al., 1983). Electroblotting: The electrophoretic transfer of macromolecules from

chromatographic gels to immobilizing matrices. Golden blot: The process of analyzing a blot with a colloidal gold probe (Brada and

Roth, 1984). HRP: Horseradish peroxidase, an enzyme commonly used in enzyme-linked assays. Hybridization: A term derived from nucleic acid analyses in which the probe binds to

the immobilized DNA or RNA via the hydrogen bonds of specific base pairing. It is sometimes misused in referring to the binding of proteins or ligands to blotted proteins (e.g., antibodies with their respective antigens).


TABLE I ( c a t i n u c d )

Immunoblot: The process of analyzing a blot with an antibody for the purpose of detecting an antigen.

Immunogold Colloidal gold associated with an antibody or S. aurm protein A to be used as a detecting reagent for the ltxalization of an immobilized antigen.

Lps blotting: Transfer and imrnunodetection of lipopolysaccharides (Bradbury et al., 1984).

Native blot: The process of transfemng proteins from isoelectrofocusing gels (Reinhart and Malamud, 1982a).

NC: Nitrocellulose membrane filter. N6rthem blot: RNA blot. Overlay: The process of probing a blot, that is, incubating a blot in a solution

containing a probe. Originally derived from assays in which the gel was maintained stationary and horizontal and actually covered with a minimal layer of fluid containing a probe.

PCM: Rehybridization: see quench. Quench

Positively charged nylon membrane filter.

The process of sequestering or blocking all unoccupied potential binding sites of an immobilizing matrix for the purpose of preventing nonspecific background.

Replica: A transferred immobilized electrophoretic pattern, that is, a blot. Southern blot:

M. Southern (1975) describing the technique of the analysis of blotted DNA from agarose gels.

immobilized flies (Tchen et al., 1985).

(Lanata et al., 1985).

overlay of protein blots (Reading and Hickey, 1985).

DNA blot. The term was originally derived from the publication of E.

4 w h blot: The process of analyzing the DNA or RNA content of squashed

Stool blot MM~: The DNA analysis of bacterial colonies derived from stool samples

WELLA: Western enzyme-linked leain analysis, that is, enzyme-conjugated lectin

Western blot: Protein blot (Burnette, 1981). Vacuum blot: The process of accelerated transfer of proteins from gels to

immobilizing matrices by employing a vacuum (Perferoen et al., 1982).

or nonionic detergents. Quenching is followed by reacting the blot with the probe, in this case the antibody. The incubation (1-2 h) is normally performed in a quenching buffer. Then the blot is washed in buffer and reacted with a labeled second antibody in quench solution. The second antibody may be radioiodinated or enzyme-linked. Staphylococcw aurew protein A has also been extensively used in place of the second antibody. After the blot has been washed, thus removing the unbound second antibody, the antibody-antigen complex can be detected. When radiolabeled ligands are used, autoradiography is employed. Should the second antibody be enzyme-linked, the blot is incubated in a solution containing the corresponding substrate to give a colored precipitate at the position of the immobilized antigen.


In such procedures nanogram levels of antigens can be detected. Quite obviously, there are many variables that can influence the quality of the final result. Before dealing with some of these, the issue of whether the system being studied is amenable to blot analysis must be addressed.

IV. TO BLOT OR NOT TO BLOT.. . THAT IS THE QUESTION

Often the final goal of a specific project is “crystal clear” and one can envision the use of protein blotting as the ultimate means for proving a point. However, translating the anticipated “figure” into reality is sometimes problematic. This may be due to incompatibility between the blot procedure used and the system being studied. Preliminary tests should be conducted to determine working conditions before a complete blot experiment such as that described above is attempted. The impor- tance of this step cannot be overemphasized. This is especially true when novel assays are being developed or new reagents are being introduced.

The simplest means for determining initial conditions is to do a series of dot blots (Hawkes et al., 1982). In such assays, droplets of samples are directly applied to an immobilizing matrix. This is then processed through quenching, probing, and detection. Based on the signal-to-background ratio obtained and the specificity of the signal, one can determine the optimal conditions to be used when blotting a gel. A general dot-blot protocol is given in Section XIV. 1. The use of dot blots as a first step preceding gel blotting is a common practice (e.g., Glenney et al., 1983; Vissing and Madsen, 1984; Gershoni et al., 1985a), and considerable information can be gained by carefully designing a well controlled experiment.

1. Quantitative Dot Blotting

A corollary to the simple dot-blot procedure is to apply serial dilutions of a sample to one filter. This approach enables the determination of detection limits for a particular protocol. Thus, even when using an enzyme-linked probe that generally renders qualitative information, one can obtain fairly quantitative results by gauging the range of dilutions used. This simple assay can be surprisingly effective (see, e.g., Leary et al., 1983; Hsu, 1984; Jahn et al., 1984; Gershoni et al., 1985a, 1986; Handman and Jarvis, 1985; Kumar et al., 1985; Nakamura et al., 1985).


2. Dot Blotting as a Screening Assay

Often the objective is to screen a series of samples, aliquots, or fractions against one probe, or various probes against one protein sample (see Glenney et al., 1983; Littauer et al., 1986). For this, a number of commercial devices have been produced. In principle, they consist of a manifold that allows actual vacuum-mediated filtration through the immobilizing matrix. A large piece of immobilizing matrix (-9 X 15 cm) is placed into a Plexiglas manifold, which forms well-separated filtration chambers. Some machines have the appearance of a typical 96-well tissue culture plate. The manifold may be used for the application step, each “well” for a different sample. Afterward the dotted filter is removed from the manifold and processed as described in Section XIV. 1. Alternatively one can treat each sample separately, retaining the filter in the assembly and performing the different washes, and so on, by vacuum fdtration through the matrix. Manifolds can be extremely useful for the application of relatively large volumes of dilute samples to a discrete “spot.”

In summary, dot blotting should be a first approach for testing the suitability of blotting for the particular question on hand. By modifying the conditions and designing a well-controlled assay, dot blotting might even be found to be sufficient in itself. If, on the other hand, gel transfers are required, the gel system and how it can affect blotting should be considered.

V. GELS

There are a number of aspects regarding the type of gel and its composition that can influence a blot experiment. Some of these are discussed in this section.

1. Sample Preparation

Once a specific signal has been obtained in a dot-blot experiment, one may want to ascribe the binding activity to a discrete component of the protein mixture. This requires resolution of the protein sample into its constituents, a matter conveniently accomplished by gel electrophoresis. Often, the process of solubilizing the sample may already be detrimental. The mere dissociation of subunits disrupts quaternary interactions that could be crucial to the maintenance of functional or recognizable configurations (see Islam et al., 1983; Thorpe et al., 1984). It is advisable


to use conditions under which the protein sample is denatured as little as possible. Unfortunately, one cannot always run nondenaturing gels, and most often, efficient resolution of membrane proteins is achieved only in the presence of anionic detergents (e.g., SDS). Moreover, reducing reagents such as 2-mercaptoethanol or dithiothreitol are also commonly required for optimal separations as they prevent aggregation via interchain disulfide bridges. Therefore, a positive dot-blot experiment does not necessarily promise successful overlays of gel transfers.

The components of sample buffers (i.e., SDS, 2-mercaptoethanol, EDTA, urea, etc.) should be tested for their individual and joint effect on the proteins being studied. This can be accomplished by running dot blots. Protein samples are suspended in various concentrations of the different sample buffer constituents. These samples are then applied as dots to a blotting matrix and tested for their ability to bind the probe. In this way optimal conditions for sample preparation can be established. One should be aware, however, of the possible effects perturbants may have on the adsorption of the “dot” to the filter. This obviously would affect the intensity of the signal, not because of loss of essential structure but rather because of reduced amounts of protein present on the filter. This last issue can be monitored by using radiolabeled protein to test the effect of various reagents on the adherence of the protein to the filter.

2. Acrylamide Concentration

The gel itself may affect the final results of an experiment in a number of ways. The concentration of acrylamide and cross-linker will dictate the dimensions of the pores through which the proteins must migrate. The denser the gel, the more difficult it will be for proteins to be eluted (DuBois and Rossen, 1983; Gershoni and Palade, 1983). Practically speaking, this becomes appreciable only for high molecular weight (> 90 kDa) proteins. Running “gradient gels” can help in this respect because more efficient elution for high molecular weight proteins is achieved in the areas of low acrylamide concentration, while reasonable resolution of low molecular weight proteins is ensured by the ever- increasing gel density. Other approaches have involved the use of reversible cross-linkers in the gel (Renart et al., 1979; Tas et al., 1979; Bolen et al., 1982), composite agarose-polyacrylamide gels (Elkon et al., 1984), protease nicking of the high molecular weight protein during blotting (Gibson, 1981), or introduction of low concentrations of SDS in the transfer buffer (Erickson et al., 1982; Nielsen et al., 1982).


3. Transfer of Fixed Gels

The question of how to preserve a gel before blotting sometimes arises. In fact, the easiest solution to this problem is to blot immediately after gel electrophoresis. Yet, if for some reason one needs to delay blotting for a time, this too is possible.

For short periods of time (up to 2 h), leave the gel in its glass plates and store them at 4°C. Should gels need to be stored for substantially longer periods, it is advisable to fix them until blotting can be performed. This is achieved by placing the gel in a “destain” solution (eg., 25% isopropanol : 10% acetic acid in water), which will preserve the electrophoretic pattern indefinitely. Just before blotting, the gel is incubated in transfer buffer until it completely reaches equilibrium with the alkaline pH. Washing (3-4 x 10 min) and shaking in a reasonable volume (100-200 ml) of transfer buffer is usually sufficient. Then blotting proceeds as normal.

Should the transfer efficiency be markedly reduced, further equilibration in transfer buffer may help. On the other hand, fixation tends to precipitate the protein bands and also removes SDS. Some proteins may not be sufficiently negatively charged upon equilibration, a problem easily solved by introducing 0.1 % SDS to the equilibration solution.

From the discussion above it is clear that Coomassie brilliant blue stained and destained gels are amenable to blotting (Jackson and Thompson, 1984; Jackson et al., 1985). In such cases the stained proteins are efficiently transferred and retained on the immobilizing matrix, and a Coomassie brilliant blue pattern on the nitrocellulose sheet is observed. One ought to be aware that the dye is eluted off the protein bands much quicker than the proteins themselves. Thus the dye pattern should not be taken as an indication of transfer efficiency. In addition, the presence of the dye may interfere with subsequent analyses (Lin and Kasamatsu, 1983). If this is the case, Coomassie brilliant blue stained patterns can be decolorized with dimethyl sulfoxide prior to blotting. Interestingly, Perides et al. (1986) have reported that fixed, stained, and dried gels can still be blotted; however a 50% drop in efficiency compared to fixed gels is observed.

The transfer of silver-stained gels is a bit more complicated. Silver staining is not always run to completion; thus a particular band may be only partially stained. That fraction which is not complexed with silver will electroelute, while the “stained” portion will remain fixed in the gel (N. Reiss, Weizmann Institute, personal communication).


4. Isoelectrofocusing and Two-Dimensional Gels Two special cases of gels that have been used for blot analyses are one- and two-dimensional gels in which isoelectrofocusing has been performed. The subject of blotting two-dimensional gels has been extensively reviewed (Symington, 1984; see also Anderson et al., 1982). Generally speaking, when the second dimension is performed in SDS, the final two-dimensional gel can be regarded as any other SDS gel. Gradient electric fields should be used in electrotransfer of two- dimensional gels only when one of the dimensions is based on differ- ences of molecular weight; otherwise a uniform electric field should be employed (see Section VII.2.A.a).

For isoelectrofocusing gels, and agarose gels in general, it has been found that the preferred modes of transfer are by diffusion and by capillarity (Reinhart and Malamud, 1982a,b; Grace et al., 1985; Hand- man and Jarvis, 1985; Hoffman and Jump, 1985). Efficient transfer can be achieved by simply applying a filter to the agarose or acrylamide gel and stacking Whatman filter paper and a weight onto the gel/ immobilizing matrix assembly. Attempts to remove the agarose gels from their plastic supports to allow electrotransfer seems to be more work than is necessary and of no advantage. This is especially true in light of the fact that efficient transfer by diffusion is possible within an hour or two. Nishizawa et al. (1985) have used specially reinforced polyacrylamide gels, which allow subsequent electroblotting without the need to remove the mechanical support.

VI. THE IMMOBILIZING MATRIX

The process of blotting entails the transfer of macromolecules to an immobilizing matrix. Matrices can be divided into two general types, microporous membrane filters and cellulose-based papers (see Press- wood, 1981). The most commonly used material is nitrocellulose membrane filters (NC), and probably this should be taken as a first choice in setting up a blotting experiment.

There are a number of factors that can determine the suitability of one matrix over another and they should be considered.

1. Chemical Composition

A preferred matrix should adsorb the protein samples efficiently and bind the subsequent probe as little as possible. The first requirement


relies on the chemical or physical interactions between the protein and the filter medium, the second is achieved by quenching (see Section IX).

Most proteins at pH values above 7 are negatively charged. It is therefore surprising that NC (also negatively charged) is such an efficient blotting matrix. Clearly hydrophobic interactions play a major role in the adsorbance of protein to NC. This is demonstrated by the fact that nonionic detergents (e.g., Triton X-100) are quite effective in removing bound protein from NC (Gershoni and Palade, 1982; Kakita et al., 1982; Lin and Kasamatsu, 1983; Flanagan and Yost, 1984). This fact should be taken into consideration when incubation and washing solutions are designed, since the presence of excessive amounts of detergent can remove up to 80% of the bound protein. Fixing blotted molecules to NC can, of course, help overcome the loss of this protein during processing. AlcohoVacetic acid, as well as glutaraldehyde, have been used (Gershoni and Palade, 1982; Jahn et al., 1984; Faye and Chrispeels, 1985). Chemical cross-linking (Kakita et al., 1982) and ultraviolet irradiation (Cannon et al., 1985) have also been employed.

Another consideration that should be taken into account in choosing a matrix is the amenability to selective staining of the transferred protein pattern. NC seems to be the easiest filter to deal with in this respect, whereas the nylon-based membranes (e.g., Zetabind, Zetaprobe, Nytran, and Genescreen) are problematic with regard to stainability (see Section VIII).

The main drawbacks of NC are its moderate binding capacity for protein (-80-100 pglcm2) and the reversibility of this association. It should be noted that not all “ N C fdters are pure nitrocellulose, and some manufacturers incorporate cellulose acetate in their NC products (e.g., the Millipore HAWP series). This modification decreases the protein-binding capacity even further. Therefore, other media have been used, which have better binding characteristics.

Alwine et al. (1 979) introduced diazo-modified cellulosic papers (DBM and DPT papers; see Table I). These filters offer a covalent linkage of the transferred protein to the paper (Renart et al., 1979; Reiser and Wardale, 1981; Renart and Sandoval, 1984). This would appear to be a significant improvement, especially since the reactive side group can be chemically quenched after transfer. However these papers are a bit more awkward to use compared to NC. First, only a precursor of such papers, the amino derivative, can be stored. The filters are then activated by nitric acid treatment just before blotting. Because the diazo groups are labile and highly reactive with amine- containing molecules (e.g., glycine), transfer must be performed quickly


(within a matter of hours) and usually in borate or phosphate buffers (Bittner et al., 1980; Olmsted, 1981; Reiser and Wardale 1981; Sym- ington et al., 1981; Renart and Sandoval, 1984). This last requirement demands that most "Laemmli" gels (Laemmli, 1970) be preequilibrated in borate or phosphate buffers to remove the otherwise reactive Tris and glycine. Regrettably, the binding capacity of the diazo papers is not significantly different from that of NC. When covalent binding is important, the diazo papers are useful. This has been shown by Olmsted (1981), who used blotting as a means for purifying monospecific antibodies (see Section XIII. l.A). Cyanogen bromide papers (Clarke et al., 1979), which also provide covalent immobilization, have been used for blotting as well (Newman et al., 1981).

Positively charge-modified nylon-based membrane filters (PCM) have been introduced as an efficient blotting matrix because they have an exceptionally high binding capacity (>500 pgkm') and practically irreversible binding; moreover, the filters require no preactivation (Gershoni and Palade, 1982). However the attributes of PCM turn out to be its main drawback. Efficient quenching of these filters is sometimes difficult to achieve. It has been found that relatively high concentrations (5- 10%) of quenching materials, and elevated temperatures (40-50°C) may be required to reduce nonspecific background. Moreover, most dyes that stain protein (e.g., Coomassie brilliant blue, Amido black, or Ponceau S), stain these filters equally well, so destaining becomes impractical. Nonetheless, PCM has been found to be extremely useful where reliable quantitative blotting is necessary, and especially where the probe is itself positively charged (e.g., Gershoni et al., 1983). For example, the ''Ru overlays of dot blots shown in Figure 1 are practical only on PCM. In addition, the analysis of small peptides appears most efficient with PCM. There have also been instances of specific proteins not being adsorbed by NC, and in such cases use of PCM is absolutely essential (Miribel et al., 1986). As experience in working with PCM is increasing, the technical problems associated with it are being solved. Milk solutions (15-20% in buffer) seem to be rather effective for quenching PCM, and a number of protein stains compatible with nylon have been developed (e.g., Kittler et al., 1984; Kumar et al., 1985; Moeremans et al., 1986). It is noteworthy that PCM has been found to be an excellent matrix for nucleic acid blotting.

Other matrices that have been used are cellulose acetate (Schaltman and Pangs, 1980), and most interesting are the modified glass fiber filters (Vandekerchkhove et al., 1985; Aebersold et al., 1986), which have been developed to allow microsequencing of blotted bands.


Fig. 1. 8 6 R ~ overlays of dot blots. Membrane preparations of porcine kidneys not treated (A, C, D) or treated with ouabain (B), were directly applied to PCM (A, B) or NC (C, D). The filters were then quenched in 2% BSA in PBS (D) or not quenched at all (A, B, C). All four filters were incubated 5 min in a solution containing 8 6 R ~ and then washed in icetold buffer and autoradiographed. Note that a signal could be detected in the unquenched PCM and that this signal could be prevented by preincubation of the protein with ouabain, indicating that the nature of the signal is the result of =RU occlusion by the NalK ATPase. No signal was obtained when using NC filters even after quenching. (Courtesy of M. Shani and S. Karlish, Department of Biochemistry, The Weizmann Institute of Science.)

2. Physical Characteristics

Two main characteristics are especially important when considering a blotting matrix: the mechanical durability and the matrix density (i.e., pore size).

The matrix must undergo the manipulations, washes, incubations, and other processes without deteriorating. In this respect the cellulosic papers are most fragile, NC is quite acceptable, and PCM is excellent. Generally speaking, in routine blot experiments NC presents no special problem; however, PCM is particularly useful if a blot must be probed a number of times (see Section XIII.5). This becomes a significant consideration in DNA blots.

The porosity of a matrix is an important characteristic in determining the filtration qualities of a medium. However, in blotting it is simply required that the matrix be porous so that it can be saturated with buffer and will allow the necessary flow of current or fluid for electro- and convection blotting, respectively. More important is the reciprocal of porosity (i.e., matrix density). The more medium present per unit area, the higher the binding capacity. Thus by using filters of smaller pore size (i-e., higher matrix density), one improves the binding characteristics of the filter.

It has been prescribed when studying low molecular weight proteins to use filters of smaller pore size, thereby increasing the probability of entrapping the proteins in the filter rather than allowing them to flow through (Burnette, 198 1 ; Lin and Kasamatsu, 1983).

In procedures that actually exploit the filtration characteristics of the matrix, as in the use of dot-blot application manifolds (Section


IV.3), pore size becomes critical. Use of large pore size filters can be advantageous in such cases, reducing the extent of filter clogging (C. Gitler, Weizmann Institute, personal communication).

In summary, a number of different blotting matrices have been used. The diazo papers and nylon membranes have been introduced because of their tenacious association with the transferred proteins. Nylon has also been found to be extremely durable. Nonetheless, NC is probably still the most popular all-purpose medium available.

VII. PROTEIN TRANSFER

Once the gel has been run and the immobilizing matrix selected, it is necessary to transfer the proteins from the gel to the matrix. This can be achieved in a number of ways, the most common being electroblotting. However, diffusion blotting and blotting by convection have also been used successfully and should be considered.

1. Diffusion and Convection Blotting

If a gel is left to itself, the polypeptides start to diffuse out of it. This process is slow and proceeds in all directions. Exploiting this phenomenon, Bowen et al. ( 1980) performed bidirectional blotting generating two gel replicas by simply securing NC to either side of a slab gel and allowing this assembly to sit submerged in a buffer solution for 36-72 h. The subsequent blots obtained were identical and were then used in a routine overlay experiment (using DNA, RNA, and protein as probes). Such blotting by diffusion has also been used routinely by Hayman and others and the blots were then probed with intact cells (Hayman et al., 1982, 1983, 1985; Dion and Pomenti, 1985). The advantages of diffusion blotting are that no special equipment is required and that two identical replicas are obtained. The efficiency of elution is about 50-70% for the average protein; yet this value must be divided by 2, to determine the maximal amount of protein present on either filter. In view of this relatively low inherent transfer efficiency and the long blotting time, other means for blotting might be more advantageous.

Unidirectional blotting can be achieved using Southern’s original approach (Southern, 1975). In such a case the gel sits in a buffer reservoir (often a “puddle” of buffer) and the matrix is placed on the gel so that no contact is made between the buffer reservoir and the matrix. Pieces of blotting paper (adsorbent paper towels or Whatman chromatographic paper) are then piled on top of the matrix and topped


with a weight (often the Merck Index or Handbook of Chemistry and Physics). Buffer is drawn from the reservoir through the gel and filter due to the capillarity of the absorbent paper. This movement of fluid serves as a unidirectional driving force, purging the protein out of the gel and onto the filter.

Whereas diffusion and convection blotting are not popularly used for protein transfer from acrylamide gels, these procedures have been found to be very efficient and actually are preferred over electroblotting for isoelectrofocusing gels (see Section V.4).

2. Electroblotting

The concept of electroblotting first described for DNA transfers by Arnheim and Southern (1977) has been widely employed only since Towbin et al. (1979) and Bittner et al. (1980) described its use for protein transfer.

In principie, a geYfilter assembly is placed into an electric field perpendicular to the plane of the slab gel. The field exerts on the polypeptides an electromotive force that drives them out of the gel and onto the immobilizing matrix. A number of factors may affect the quality and efficiency of the blot obtained. The most relevant are the design of the electroblot apparatus, the buffer composition, and the power source used.

A. APPARATUS DESIGN

Many designs for blotting apparatus have been published (see Table I1 for some examples). An efficient machine should be so designed that the gel is subjected to a reproducible and uniform field. Obviously, a distorted, uneven electric field will exert variable forces on different areas of the gel. This can cause differential elution efficiencies of proteins, making comparisons problematic.

Most apparatus consist of a Plexiglas tank with platinum wire electrode arrays mounted near the walls of the tank. The gel assembly is placed in a cassette, which is inserted between the electrodes. Semidry electroblotting has also been made possible, and in such systems the geYfilter assembly are directly sandwiched between graphite electrodes (Kyhse-Andersen, 1984; Bjerrum and Schafer-Nielsen, 1986). Figure 2 is a detailed blueprint for a blot apparatus used in my laboratory.

Electrode Design. The geometrical design of electrode arrays has been discussed (Bittner et al., 1980; Gershoni et al., 1985b). The number of wire stretches of platinum for each electrode should be the same,

a.


TABLE I1

Designs for Blot Apparatus

Reference Electrodes Comments

Bittner et al. (1980) Platinum wires Uniform electric field Manabe et al. (1984) Platinum wires For micropolyacrylamide gels

Kyhse-Anderson (1984) Graphite plates Semidry transfer, horizontal

Stott et al. (1985) Graphite plates Uniform electric field Svoboda et al. (1985) Anode: surface conductive Horizontal orientation;

glass anodic and cathodic buffers are separated

and multiple blots

orientation

Cathode: stainless steel plate

and their positions should be such that the wires of the anode and the cathode are aligned. If one continuous wire is weaved back and forth to create an array, the cross-connecting stretches should be insulated. This can be achieved by simply mounting the platinum wire on a sheet of Plexiglas. The main stretches are aligned on the side facing the gel cassette, and the cross-connections are pulled through and run along the back side of the Plexiglas support. Separate wiring for each strand of platinum provides the possibility for a gradient electric field (Gershoni et al., 1985b). This is achieved by placing resistors between the wire and the power source. As the resistance is increased, the potential falling on the wire drops. A scheme for the production of a gradient field is given in Figure 3.

The advantages of gradient electric fields are threefold. First, one can apply a high electric field at the top of a gel where the high molecular weight proteins are resolved. This provides a means to preferentially exert more force for eluting the proteins that are otherwise less efficiently transferred (Burnette, 198 1; Howe and Hershey, 1981; Lin and Kasamatsu, 1983). Second, the low molecular weight proteins “see” a moderate or weak field sufficient for their elution, yet mild enough to prevent overacceleration, which can lead to their loss through the filter (Gershoni and Palade, 1982; Lin and Kasamatsu, 1983). Third, the gradient makes better use of the power supply: the high intensity at the top is generated at the expense of the energy saved at the bottom. This allows efficient transfer without running into high electric currents, which otherwise would heat the system.

The Cassette. The function of the cassette is to provide mechanical support for the gel/filter assembly. The cassette described in Figure

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Fig. 3. “Resistor box” for electroblot apparatus. This box contains four 5-pin connectors, two modes for each electrode. When only short-circuited connectors are used, a uniform electric field is generated. A gradient electric field is provided when the resistors are introduced. A maximal gradient is formed when both the anode and the cathode are connected via the resistors. An intermediate gradient can be formed by employing only one set of resistors and short-circuiting the other electrode.

2 is designed to be multipurpose yet simple. A Plexiglas frame is strung with surgical silk like a tennis racket (nylon fish line can also be used). As long as the mesh is taut, the gel/filter will be well supported. The advantage of this design is that the silk presents no appreciable electrical resistance as it becomes saturated with the buffer (use of nylon will create only negligible resistance). In commercially available apparatus using perforated plastic sheets, the cassette can cast a “shadow” on the gel, which interferes with transfer efficiency (Fig. 4).

In principle, cassettes should be placed midway between the electrodes, since this is the most uniform plane of the electric field (Gershoni et al., 1985b). As one gets closer to the electrode wires, hot spots in their vicinity become more apparent. Therefore, the simultaneous transfer of two or more gels, by using a number of cassettes placed in different planes of the apparatus, should not be attempted. This usually leads to different and uneven transfer efficiencies for each filter. Transferring more than one gel at a time can be accomplished by making multilayer gel/filter assemblies (e.g., Scotch Brite/geYfilter/ Scotch Brite/gel/filter/Scotch Brite, etc.).

20 JONATHAN M. CERSHONI

Fig. 4. The effect of nonconductive cassettes. Radioactive BSA was introduced into a solution of polyacryiamide prior to polymerization. Thus a gel was prepared that contained a uniform distribution of labeled protein. This was then blotted to NC in an apparatus equipped with a perforated plastic cassette. After blotting, the NC was autoradiographed. Clearly the pattern of the autoradiogram (B) corresponds precisely to the perforations of the cassette (A).

B. TRANSFER BUFFERS

The vast majority of SDS-polyacrylamide gels run today use the "Laemmli buffer system" in which the gel to be transferred contains 25 mM Tris, 192 mM glycine, and 0.1% SDS (Laemmli, 1970). Transferring in this buffer is feasible; however, the conductivity is rather high (> 700 Fmho) and therefore high currents are generated even at low voltage values (Fig. 5). Moreover, the presence of SDS affects the binding of protein to NC. The transfer buffer should therefore be of relatively low ionic strength and should not contain SDS. In such cases, however, the polyacrylamide gel may swell. This problem can be mitigated by introducing methanol into the transfer buffer, as was originally suggested by Towbin et al. (1979) [isopropanol has also been used (Clegg, 1982)]. Alternatively, preswelling of the gel is possible by equilibrating the gel in transfer buffer before blotting. Neither of these approaches is ideal. The use of methanol-containing buffers will render elution less efficient.

Transferring in buffers lacking methanol may be more effective with regard to elution, but some of the eluted proteins may not be well adsorbed by the matrix [a problem particularly noticeable for low molecular weight proteins (Gershoni and Palade, 1982) and for specific proteins such as calmodulin (Van Eldik and Wolchok, 1984; Rochette-


i r i s concentration ( ~ H J

L 5 300

100

I I I I

100 200 300 LOO 500

Conductivity IpMHO)

Fig. 5. The effect of salt concentration on buffer conductance. A solution of 25 mM Tris was titered with glycine to give a pH of 8.3. This was then poured into a blot apparatus, which was operated either with a uniform electric field (constant 50 V) or with a gradient electric field (in which the uppermost electrode pair was kept at a constant 50 V). The current was measured as the buffer was progressively diluted. Note that the absolute conductivity of the buffer is also provided.

Egly and Daviaud, 1985)l. Once again, the use of a gradient electric field may overcome this problem, since the low molecular weight proteins have more time to interact with the immobilizing matrix. Other solutions include using NC of a higher matrix density (Burnette, 1981) or simply transferring to PCM (Gershoni and Palade, 1982).

c. THE POWER SOURCE

When considering electrotransfer, the question of “constant current versus constant voltage” may arise. Basically, one should use constant voltage because it is the potential difference the proteins “see” that drives them out of the gel. As transfer proceeds, however, the electro- lytes in the gel are also eluted and contribute to the conductivity of the buffer. This results in a drop of resistance, demanding an increase in current to maintain constant voltage. Currents greater than a few hundred milliampere result in considerable Joule heating of the system, which further reduces the resistance. It is therefore advisable to try and maintain transfer currents under 300 mA. Employment of gradient electric fields and low ionic strength buffers are useful approaches.


Precooling the transfer buffer or running the transfer in the cold room is also helpful. Commercial apparatus usually provide cooling coils or allow the buffer to be circulated through a cooling system. These options, however, may be found unnecessary as long as currents are kept low.

In any case, the matter of a special power supply for blotting arises only when currents higher than 200-300 mA are anticipated. This is because most power supplies designed for gel electrophoresis are built to provide high voltage and moderate currents. When using such power supplies for blotting, it is most efficient to run at constant maximal current. In this way the capacity of the power source is never exceeded. Recently, a number of power supplies for blotting have been introduced into the commercial market and they obviously are designed to generate high currents (2-5 A), thus enabling one to blot while maintaining constant voltage.

The time required for efficient blotting is different for each protein being transferred (see, e.g., DuBois and Rossen, 1983). High molecular weight polypeptides blot slower than do low molecular weight proteins (Burnette, 1981; Howe and Hershey, 1981; Lin and Kasamatsu, 1983). Generally electroblotting should proceed for at least 2 h, but more time may be required depending on the buffer conditions, the type of gel used, the field intensity, and the molecular weight of the proteins to be eluted. Some protocols prescribe 22 h of blotting as a routine (Burnette, 1981). In addition, some proteins may be particularly “stubborn.” Basic proteins are eluted most efficiently in high-pH buffers (Szewczyk and Kozloff, 1985).

In summary, there are many factors that affect the final gel replica. When possible, it is preferable to use a gradient electric field (50-10 V, 200 mA) and a low ionic strength buffer with no methanol. Under such conditions reasonably efficient transfers can be achieved in a matter of 2-3 h.

VIII. STAINING OF BLOTS

The visualization of the transferred protein pattern immobilized on the matrix can be useful in evaluating the quality of transfer and, more important, to allow the correlation of the eventual signal to a specific band. A number of approaches have been adopted for blot staining, but the vast majority are restricted to NC blots.

The simplest staining procedures entail incubating the blot in an alcohollacetic acid solution containing dye [e.g., Amido black (Towbin


et al., 1979), Ponceau S (Muilerman et al., 1982), Coomassie brilliant blue (Burnette, 1981)]. Staining is followed by destaining in the same solvent minus the dye. Amido black tends to be rather sensitive [a 25 ng dot of bovine serum albumin (BSA) is easily detected] and acceptable background can be achieved. Coomassie brilliant blue tends to give higher background; Ponceau S gives very clean patterns, but it is slightly less sensitive than Amido black. None of these dyes can be used for nylon-based membranes because the background is as high as the signals and destaining is not possible. The choice of solvent can alter the quality of the blot. Methanol, for example, tends to be more destructive for NC than is isopropanol. Therefore it is suggested that staining and destaining be performed in 25% isopropanol/lO% acetic acid (Gershoni and Palade, 1982). Ponceau S is readily water soluble; staining in 7% trichloroacetic acid and destaining in isoproponal/acetic acid provides a permanent stain, while a transient pattern can be obtained by destaining in water (excessive wash in water eventually removes not only the background but the signal as well). Transient staining can be useful because it allows the visualization of a pattern that can subsequently be used for probing without the concern that the presence of the dye may have an effect on the results. Toluidene blue has also been used in this manner (Towbin et al., 1982). Another way to visualize the transferred pattern while maintaining the proteins unaltered is to “negative-stain” the blot. This is accomplished by incubating the blot in a dilute enzyme solution followed by histochemical detection of the bound enzyme (see Section XIV.4). Use of Tween 20 as a quench provides the option of staining the blot after it has been probed (Batteiger et al., 1982).

To obtain a protein pattern for nylon-based membranes, the transfer of prestained proteins is also possible (Jackson and Thompson, 1984; Lubit, 1984; Falk and Elliot, 1985). Proteins stained prior to electrophoresis can be used as standards to be run in parallel to the lanes of the samples being tested. Another possibility is to stain the gel with Coomassie brilliant blue after electrophoresis and to transfer the stained gel (see Section V.3). Obviously, blotting radio labeled standards can always provide an internal reference to be detected upon autoradiography .

This brings us to the question of highly sensitive stains for blots. Regrettably, silver stains on blots have been of limited use. Usually, one obtains a negative picture; that is, the background is dark and the protein bands remain unstained (Yuen et al., 1982). Recently, efficient and sensitive silver stains have been developed that are compatible with both NC and PCM (Merril and Pratt 1986) and these will undoubtedly


be extremely useful. One of the most sensitive stains has been achieved with colloidal gold; for examples see Brada and Roth (1984), Hsu (1984), Moeremans et al. (1984,1985), Surek and Latzko (1984), Daneels et al. (1986), and Rohringer and Holden (1985). These procedures are very sensitive and are reasonable to perform. PCM is not amenable to colloidal gold procedures due to excessive background staining. Iron staining of nylon-based membrane blots was suggested when these membranes were introduced as blotting matrices (Gershoni and Palade, 1982). An improved procedure has been developed using colloidal iron, which is more sensitive than Coomassie brilliant blue staining of gels yet not as sensitive as the colloida! gold staining of NC blots (Moeremans et al., 1986).

Another approach for staining blots has been to chemically modify the immobilized proteins with a hapten and subsequently detect the latter with an antibody specific for it (Wojtkowiak et al., 1983; Kittler et al., 1984; J. M. Wolff et al., 1985). Such procedures are routine immunoblot assays for the detection of hapten-modified proteins. These stains have also been found suitable for the nylon-based membranes. Biotinylation of proteins on the blot provides a sensitive general stain (LaRochelle and Froehner, 1986) or the selective detection of specific proteins such as sulfydryl-containing polypeptides (Bayer et al., 1985; Roffman et al., 1986) and surface membrane proteins (Hurley et al. 1985).

An additional stain has recently been developed in which the immobilized proteins are iodinated with nonradioactive iodine, which is then detected with a starch-containing solution to give a purple signal (Kumar et al., 1985). India ink has also been used for the staining of NC blots (Hancock and Tsang, 1983).

Intensification of stained patterns is also possible. Silver staining of colloidal gold patterns enhances the signals obtained (Moeremans et al., 1984). Nickel and cobalt enhance horseradish peroxidase reaction products (e.g., De Blas and Chenvinski, 1983).

In summary, numerous procedures have been developed to visualize a blotted pattern. Staining NC filters is easily accomplished using standard protein stains such as Amido black. More sensitive stains are based on the use of colloidal gold. Colloidal iron has been used for the nylon-based membranes. Immunodetection of hapten-derivatized proteins can also provide a general protein stain.

IX. QUENCHING

After protein transfer, the blots are probed. However, because the probe itself can bind nonspecifically to the immobilizing matrix, an


intermediate step must be introduced, namely quenching. This is the process of blocking all unoccupied binding sites of the filter.

Quenching is accomplished by incubating the blot in a buffer solution containing a “quencher,” most often an “inert” protein (e.g., BSA, hemoglobin, gelatin, fetal calf serum, milk). The requirements of an efficient quencher are that (1) it sequesters all unoccupied binding sites remaining on the filter, (2) it does not remove the transferred bound proteins, (3) it does not obscure or “inactivate” the protein to be probed, and (4) it does not interfere with the probing process. Other demands might be that the quencher be cheap and accessible in a dependably reproducible composition-for example, the variability between different lots of calf serum may be a problem.

It is advisable to optimize the quenching of a blot, which may be critical for obtaining acceptable signal-to-background ratios. Calibration of the type and amount of quencher, as well as time and temperature of incubation, can be accomplished in a dot-blot experiment. In fact one does not necessarily need to apply a “dot” of the sample. All that is being asked is, What are the best conditions to prevent nonspecific binding? Thus quenching and probing blank filters often suffice. To the question Can one overquench? the answer is yes. Excessive quencher can remove bound protein. This can be monitored by “dotting” a sample of radio-labeled protein and counting the filter before and after the quench. Some quenchers are excellent for one assay and horrendous for others. A case in point is hemoglobin. Hemoglobin is often found to be a superb quencher that can be used in concentrations as low as 0.1% (which more than compensates for its relatively high cost), contains virtually no oligosaccharide contaminants (in contrast to BSA), is not particularly “sticky,” and has a neutral PI. However, it does have intrinsic peroxidase activity, thus rendering it mediocre for blots des- tined to be probed with horseradish peroxidase conjugates. Polyvinyl- pyrrolidone has been found to be particularly suitable for wheat germ agglutinin overlays of protein blots (Bartles and Hubbard, 1984). Milk, surprisingly, is a very good quencher (Johnson et al., 1984). A list of quenchers is given in Table 111.

An alternative approach to quenching with proteins is the use of detergents. Conceptually, detergents are not really quenchers because they do not sequester binding sites but rather reduce the nonspecific “stickiness” of the probe and filter. Most often, nonionic detergents (e.g., Triton X-100, Nonidet-P 40, and Tween 20) have been used. The use of detergents is attractive because these substances are cheap, readily available, easily handled, and rather efficient. Their major drawback is that they definitely interfere with the association of protein with the immobilizing matrix. As much as 80% of bound BSA can be


TABLE 111

Quenchers" -~ ~~~ ~~

Quencher Concentration Filter Probe Ref.6

Casein Saturated NC Low-density 7

2% NC Antibody 12 1% NC Antibody or lectin 14

Gelatin 0.25% NC Antibody 4 3% NC Lectin 8 0.1% + 1% PCM Calmodulin 9

0.2% NC Antibody 1 1 Liquid gelatin 3% NC Antibody 15 Milk 5% NC Antibody 10

5% NC DNA 13 Ovalbumin 1% NC Cells 5 Pol yvin ylpyrrolidone 2% NC Wheat germ 1

Tween 20 0.05% NC Antibody 2 0.5% NC Antibody 3

lipoprotein

hemoglobin

agglutinin

0.3% NC Antibody 6 ~ ~ ~~ ~

a This table gives examples of quenchers other than BSA, hemoglobin, Triton X-100, and Nonidet P-40, which are commonly used. ' Key to references: 1. Bartles and Hubbard (1984). 2. Batteiger et al. (1982). 3. Blake et al. (1984). 4. Bradbury et al. (1984). 5. Carnow et al. (1985). 6. Daneels et al. (1985). 7. Dresel and Schettler (1984). 8. Faye and Chrispeels (1985). 9. Gorelick et al. (1983). 10. Johnson et al. (1984). 1 1 . Lin and Kasamatsu (1983). 12. Mandrel1 and Zollinger (1984). 13. Miskimins et al. (1985). 14. Ramirez et al. (1983). 15. Saravis (1984).

removed with 0.1% Triton X-100. In this respect Tween 20 has been found to be the most acceptable detergent quencher and, in general, the concentration of the detergent should not exceed 0.2% (Batteiger et al., 1982). An added advantage to the exclusive use of a detergent is that after probing, the blot can still be stained for protein.

X. PROBING AND WASHING

The quenched blot is now ready to be probed. This simply entails incubating the blot in a solution containing a diluted probe (antibody, hormone, lectin, toxin, etc.). A rule of thumb is that one should probe in a quenching solution. The rationale behind this is to prevent nonspecific adsorption of the probe to irrelevant areas of the blot. To


conserve material, it is usually sufficient to use a lower concentration of quencher. The time and temperature of incubation, as well as the concentration of the probe and specific requirements (e.g., the presence of divalent cations), should all be determined by dot blotting.

The practical problem of actually handling the blot should be considered. Nucleic acid blots are usually quenched and probed in “seal-a-meal” bags. This practice is advantageous because it allows the use of small amounts of reagents and provides all the attributes of disposable incubation chambers. Unfortunately, protein blotting is not amenable to the use of such bags, which often lead to high nonuniform background (swirls, slurs, blotches, bald spots, and other problems). Therefore, protein blots should be incubated in comparatively large volumes of fluid so that the blot is lavishly bathed. Moreover, the solution should be well mixed, rocked or shaken (50-80 cycles per minute). Compartmentalized plastic boxes (such as those manufactured by Althor Products, Wilton, CT) are extremely useful. Rocking has the advantage over shaking in that one can reduce the volume of solutions considerably. Incubation of blots with very small quantities of “precious” probes can be achieved but requires special manipulation of the blot (Gershoni and Palade, 1982; Muilerman et al., 1982; Douglas and King, 1984).

After probing, one should rinse the blot-that is, aspirate the probe solution and replace it with a generous volume of buffer (no quencher is needed), which is immediately removed, and the process repeated a few times. Once rinsed, the filter is washed-that is, incubated in buffer with vigorous shaking for at least 5-10 min (no quencher is needed). Four or five such washes (a total of approximately an hour) is usually sufficient. When special ingredients are required for probing, these supplements should be included in all wash solutions (e.g., the presence of Ca2+ is required for calmodulin or concanavalin A overlays). When the complex between the probe and the immobilized protein tends to be labile, excessive washes can markedly reduce the signal. In such instances, washing with ice-cold buffers can be helpful, and one should increase buffer volumes and decrease the time of wash. For example, three 5 min washes are adequate for washing a-bungarotoxin overlays of protein blots (Gershoni et al., 1983).

If the background is intolerable, consider introducing nonionic detergents in the wash regimen. For example, try including one 5-10 min wash with 0.1% Tween 20 in buffer. Overuse of detergent can diminish the final signal.

For most routine overlay experiments (e.g., immunoblotting or lectin overlays in which the complex formed is stable), washing is a convenient


step to stop a protocol: leave the blot in wash solution, and go home and have dinner (what is referred to professionally as “was washed overnight”). The following morning the wash regimen can proceed, exchanging wash solutions a number of times.

Should a second probing be necessary-for example, with horseradish-peroxidase-conjugated anti-antibody-probing followed by additional washing is performed. Afterward the blot is processed to detect the complex.

XI. SIGNAL DETECTION

There are two main procedures for demonstrating protein-ligand associations: autoradiography (when the probe is radioactive) and histochemical staining (when the probe is an enzyme conjugate). These two approaches are not necessarily interchangeable and can sometimes even be used together for double staining. It seems that autoradiography is easier to control and thus is more likely to provide optimal final results. Enzyme reactions often are quicker and tend to provide better signal resolution.

1. Autoradiography

In contrast to gels, blots are thin, flat, and have no tendency to curl. Therefore, autoradiography can be performed by placing the blot between two sheets of plastic wrap (e.g., Saran Wrap). Completely dried blots can be placed directly against the film. However, drying a blot usually causes considerable irreversible denaturation of the probe, which makes its removal difficult if not impossible, thus hindering the chances for multiple use of the same blot (see Section XIII. 5). The plastic wrap should be pulled taut over the surface of the blot, since wrinkles sometimes cast shadows on the final autoradiogram. It is helpful to use a cardboard support for mounting blots before exposure. First the cardboard is covered with plastic wrap secured by masking tape; then the blot is applied and is covered by a second layer of wrap. Direct application of the blot to the cardboard can sometimes lead to artifactual blemishes, particularly on corregated cardboard, because the “ribs” soak up excess buffer from the damp blot and thus become preferentially labeled with the residual radioactive probe.

Quantification of the bound probe can be achieved by either scanning the autoradiogram (Lin et al., 1984) or excising the area containing the signal and counting it (Howe and Hershey, 1981; Batteiger et al., 1982;


Gershoni and Palade, 1982, Gershoni et al., 1983; Lin et al., 1984; Maruyama et al., 1985). In the latter situation care must be taken to excise the entire signal with as little background as possible. Another patch from the same blot, equivalent in size to the first but lacking any signal, should be counted to give a value for “background.” In this manner the net signal can be calculated. If low-energy radioisotopes are to be detected, scintillation can be used (Bonner and Laskey, 1974). The application of 2,5-diphenyloxasole (PPO) in toluene or xylene can be performed (Southern, 1975). Commercially available amplification solutions for fluorography can be used as well (Burnette, 1981; Roberts, 1985).

2. Detection of Enzyme Conjugates

The use of radioactive probes is subject to the objections that they are somewhat hazardous and need to be continually prepared because of the short half-life of ’*P or lZ5I. A reasonably sensitive, less hazardous, and very stable alternative is an enzyme-linked probe. In principle, a probe of biological binding activity is chemically linked to an enzyme that is easily detectable histochemically. Most commonly used are horseradish peroxidase and alkaline phosphatase, although other enzymes have also been employed (see Table IV).

The conjugate is used like any other probe but, after the final washes, the filter is incubated in a substrate-containing reaction mixture. Nu- merous protocols have been devised for the detection of enzymes on blots (Table IV). A common denominator to all is that the reaction product precipitates at the site of the enzyme. It can be very useful, in the development of new assays, to consult classical textbooks on histol- ogy, cytology, and histochemistry . Often these references provide excellent protocols for the localization of specific enzymes.

A common complaint with respect to enzyme-linked assays is that the contrast seems to be dramatically lost once the filter has dried, thus impairing faithful reproduction of the results. This may be true due to actual fading of light-sensitive products (e.g., diaminobenzidine reaction products in horseradish peroxidase assays), but usually the contrast can be restored by simply wetting the blot. Even greater contrast can be obtained by making the filter transparent with xylene or immersion oil and photographing the blot with backlighting [transmitted versus reflected light (DuBois and Rossen, 1983; Ramirez et al, 1983; Maruy- ama et al., 1985; Nakamura et al., 1985)l. In such instances, caution should be taken to ensure that the solvent used to render the filter transparent does not concomitantly solubilize the precipitated dye-for


TABLE IV

Enzymes and Their Substrates Used as Detectors in Blotting

Enzymes Conjugate Substrate Ref."

Acid phosphatase Streptavidin Naphtho! AS-MX 2 phosphatelfast violet B salt

bromo-4- chloroindoxylphosphate

phosphatelfast red TR salt

phosphatelfast red TR salt

blue B

Alkaline phosphatase Goat anti-mouse IgG Nitro blue tetrazoliud5- 1

H ydrazide Naphthol AS-MX 7

Rabbit anti-mouse F(ab')o Naphthol AS-MX 9

Sheep anti-mouse 1gG B-naphthyl phosphatelfast 1 1

Alkaline phosphatase, Biotin Nitro blue tetrazolium/5- 8 polymeric bromo-4-

chloroindoxylphosphate

methosulfate/ paranitroblue tetrazolium chloride

HzOz

+ cobalt chloride and nickel ammonium sulfate

HzOz

HzOz

Glucose oxidase Goat anti-mink IgG D-Glucoselphenazine 10

Horseradish peroxidase (not conjugated) Aminoeth ylcarbazolel 3

PAP Diaminobenzidine/H*O* 4

Anti-goat IgG 4-Chloro- 1-naphthol/ 5

(not conjugated) 4-Chloro- I -naphthol/ 6

Hydrazide Diaminobenzidine/HZOz 7 ~ ~

Key to references: 1. Blake et al. (1984). 2. Brower et al. (1985). 3. Clegg (1982). 4. De Blas and Chenvinski (1983). 5. Dresel and Schettler (1984). 6. Faye and Chrispeels (1985). 7. Gershoni et al. (1985a). 8. Leary et al. (1983). 9. OConnor and Ashman (1982). 10. Porter and Porter (1984). 11. Turner (1983).

instance, the diazo dyes that result from alkaline phosphatase reactions are solubilized in xylene or methylene chloride.

Controls that should be considered in enzyme-linked assays are to test for similar enzyme activity in the sample being studied and to ensure that the enzyme does not "stick" to irrelevant bands. This can be tested by probing with the unconjugated enzyme.


A final word to the wise: always check both sides of your blot, since the histochemical reaction is asymmetric with regard to the filter’s surfaces.

Quantification of histochemical reactions can be achieved in a number of ways. Naturally, scanning the blot is possible (Ramirez et al., 1933; Nakamura et al., 1985). One can excise the bands and solubilize the precipitates to be quantified spectrophotometrically [dimethylene chloride is a good solvent for fast red precipitates of alkaline phosphatase reactions and can be read at 512 nm (Y. Hiller, Weizmann Institute of Science, personal communication)]. A third approach can be to use a substance that gives a soluble product, which is then read spectrophotometrically (e.g., p-nitrophenyl phosphate for alkaline phosphatase or o-phenyldiamine for horseradish peroxidase).

In summary, autoradiography is a convenient means to detect radioactive complexed probes. The advantages of this approach are that it is probably the most sensitive and that exposures can be repeated until optimal signals are obtained. In contrast, histochemical analyses are less hazardous, but overdevelopment of signals cannot be corrected. The resolution of enzyme-linked assays appears to be superior to that of autoradiography.

XII. CORRELATING A SIGNAL TO A BAND

The final result of a blot experiment is often a single band somewhere in an otherwise blank X-ray film or filter. It then becomes necessary to ascribe the signal to a particular lane of the gel and, more specifically, to a discrete polypeptide within the electrophoretic pattern. Therefore, one must generate internal points of reference to assist in correlating the band to a defined area of the protein pattern.

Probing individual lanes separately is a foolproof approach. Yet here too remains the problem of subsequently aligning the blots with respect to each other. Some useful suggestions for solving the foregoing problems are given next.

1. Mixed Dye Fronts

Pyronin y (a pink dye) and bromphenol blue are commonly used as marker dyes in sample buffers. In contrast to bromphenol blue, pyronin y does not diffuse laterally and therefore forms well-separated pink bands at the dye front of the gel. Moreover, pyronin y tends to precipitate slightly in the stacking gel at the surface of the well. In this


manner the top and bottom of a lane are marked with a precipitate and a pink band, respectively. By alternating pyronin y with bromphenol blue, or by using pyronin y in blank wells thereby separating samples, one can easily define the limits of a specific lane. After gel electrophoresis, each lane can be severed from the gel by cutting along the line connecting the pyronin y on the top and on the bottom. The individual lanes can then be blotted and probed separately. The pyronin y adsorbs well to NC (bromphenol blue adsorbs to PCM). The blots can be realigned by matching the preserved pyronin y marks. Where a complete gel pattern is to be probed with one reagent, blotting of the entire gel to one piece of filter is advisable. Here too the use of pyronin yt bromphenol blue is useful, indicating the bottom of the gel and the limits of each lane.

2. Internal Markers

When possible, a lane of radioactive markers or prestained markers should be run to provide an internal reference point in the blot or resultant autoradiogram. In the latter case, when ‘*C-labeled proteins are used, particular attention should be given to placing the “protein surface” of the blot toward the X-ray film, since this has a profound effect on exposure time. A positive control can serve as an efficient internal standard; for example, ovalbumin serves as a convenient positive control in concanavalin A overlay experiments. Thus if one has a reliable sample whose signal is well characterized, new signals can be compared with it. When possible, probing stained filters is extremely useful. In this respect transient staining can be of help. A blot is stained with Ponceau S and destained with water (see Section VIII). The place of specific bands is marked with a pen and destaining is continued until all the Ponceau S has been removed. This provides a “clean” blot with marked reference points. Curiously, marking filters with a pencil often leaves a shadow of the markings on the autoradiogram. In addition, some inks actually adsorb the radioactive probe and in this way generate a positive signal of the markings. New England Nuclear has come out with a special phosphorescent labeling pen called Ultemit, which is excellent for producing easily detectable points of reference for auto radiography.

3. Cut Comers

Autoradiography requires that the signal correlate not only to the gel, but to the blot as well. When the background is extremely low, the precise placement of the autoradiog;am is sometimes confusing. Slight


background provides a “shadow” of the blot, which makes matching up simple. Therefore, be sure to cut filters in distinctive ways. For example, removing a corner or excising a square of filter at the corner is useful. In fact, this can have a dual purpose. One can count the amount of background radioactivity in one square centimeter of filter. The vacant area serves as a reference point, and the value of nonspecific background per square centimeter assists in gauging the time permissible for exposure.

4. Hemoglobin Quench

Among the attributes of hemoglobin as a quench is its intrinsic brown color. Often a negative pattern of white bands on a beigehed background is generated on hemoglobin-quenched blots. It might be worthwhile to develop a stained quencher to enhance this phenomenon.

XIII. APPLICATIONS

To give the reader a practical sense of how blotting can be used, a selection of different applications is described.

1. Immunoblotting

The classical application of protein blotting has become the immunodetection of a blotted antigen (for a review on this subject, see Towbin and Gordon, 1984). Most protocols entail SDS-PAGE, and electroblotting to NC. The major variability arises in the quenchers used and the types of probes employed.

Common to almost all protocols is the requirement for a second probe, an S. aurezu protein A derivative or an anti-antibody. It is possible to detect as little as 10 ng of antigen and often even less. Obviously, if specific assay conditions for your antibody have been well worked out for conventional enzyme-linked or radioimmunosorbent assays (ELISA or RIA) these should be considered as a reasonable starting point for blotting. Where such information is lacking, the following points will be useful in setting up an immunoblot assay.

(i) Use rabbit serum at a dilution of 1 : 100 or 1 : 200. A rabbit with high titer can produce serum that can be diluted 1 : 1000.

(ii) When possible, use S. aweus protein A as a second probe. It tends to be less sticky than anti-antibodies [F(ab‘)n fragments of IgG also tend to be less sticky than intact IgG]. One should remember that IgGs of different species have different affinities for S. aureuS protein A (see Table 2 in Langone, 1980).


(iii) When whole serum is used, there are often contaminants that cause high background. This is sometimes caused by genuine cross- reactivity, especially when calf serum and the like are used to quench. Preincubation of the probe with a blank quenched filter can assist in reducing the effect of interfering contaminants (0’- Rand et al., 1985). Alternatively, if, for example, a goat anti-mouse IgG is used as the second probe, it is sometimes helpful to quench the filter with goat serum. This can reduce the extent of nonspecific associations of the labeled second probe with proteins of the electrophoretic pattern.

(iv) The use of monoclonal antibodies is sometimes problematic as these highly specific probes may recognize a determinant that is particularly sensitive to the electrophoretic process itself (i.e., solubilization, detergent denaturation, reduction, heating, etc.). Preservation of determinant integrity or renaturation of specific configurations is not easily achieved. Not boiling the sample, excluding reducing agents (e.g., 2-mercaptoethanol), running gels in the cold, or reducing detergent concentrations can sometimes help in maintaining native conformations. Posttreatment of gels or blots in renaturing buffers has also been suggested (Bowen et al., 1980; Frey and Afting, 1983; Hjerten, 1983; Islam et al., 1983; Bradbury and Thompson, 1984; Mandrel1 and Zollinger, 1984; Thorpe et al., 1984). The objective of such procedures is to remove the SDS from the protein and allow its refolding (see also Sections XIII.3.A and XIII.3.B).

A. COROLLARY APPLICATIONS OF IMMUNOBLOTTING

A number of procedures have been developed in which immunoblotting is an intermediate step.

Afinity Purtfiatwn of Monospecifi Antibodies. Olmsted (1981) has demonstrated that blots can be used to purify specific antibodies. A blot is probed with a rabbit serum. Specific polyclonal antibodies associate with discrete polypeptides. The area containing the antigen- antibody complex is excised from the blot and incubated in a low-pH solution, leading to the dissociation of the antibody from the still immobilized antigen. The antibody-containing solution is neutralized and used for immunocytochemistry . This procedure has been performed successfully with all the immobilizing matrices commonly used.

Detection of Specifii Enzymes. Muilerman et al. (1982) have developed the following procedure. A blot is incubated with an excess of

a.

b.


antibody directed against a specific enzyme. Then the blot is incubated with the enzyme itself, followed by a reaction mixture for the enzyme activity. In such instances, due to polyvalency, the antibody bridges between the immobilized inactive enzyme subunit and active enzyme. This approach has also been used by Van der Meer et al. (1983).

GeneraZProtein Stain. As was discussed previously (Section VIII), haptenized proteins can be detected using anti-hapten antibodies.

c.

2. The Analysis of Glycoproteins

Protein blotting has been used for the analysis of glycoproteins in three different ways. The first has been via probing with lectins, specific sugar-binding proteins (e.g., Glass et al., 1981; Clegg, 1982; Gershoni and Palade, 1982; Hawkes, 1982; Bartles and Hubbard, 1984; De Maio et al., 1986a,b). The second approach entails in situ enzyme modification of glycoproteins (Gershoni and Palade, 1982; Bartles and Hubbard, 1984; Kerjaschki et al., 1984a,b; Rohringer and Holden, 1985; De Maio et al., 1986a,b), and the last involves a direct sugar stain, for example, enzyme-hydrazides (Gershoni et al., 1985a; Keren et al., 1986).

A. LECTIN OVERLAYS

In many respects lectin overlays resemble immunoblotting. However, the antibody is replaced by a lectin of known sugar specificity. Second probes, such as antibodies against the lectins, can be used, yet most often the lectins are themselves radio-iodinated. Enzyme conjugates have also been prepared (Moroi and Jung, 1984; Reading and Hickey, 1985). A different approach for detection is the joint use of lectins with “glycoenzymes.” Notable is the case of horseradish peroxidase with concanavalin A (Clegg, 1982): the lectin is a mannose-binding protein, whereas the enzyme is a mannose-containing protein. Therefore one can probe a blot with concanavalin A and simply reprobe with horseradish peroxidase. The presence of the bound glycoprotein is revealed by the enzyme activity. A similar approach has been used in detecting wheat germ agglutinin with avidin followed with a biotinylated enzyme (Rohringer and Holden, 1985). It would be useful to generate an arsenal of specific “glycoenzymes” to be used in conjunction with their corresponding lectins.

Lectin overlay of gels has been found to be an acceptable means for detecting glycoproteins (Burridge, 1978). Overlaying of blots, however, provides additional flexibility that cannot be realized using gels. For example, once a complex has been created and detected, it can further


be analyzed by subjecting it to solutions containing sugar haptens; thus the bound lectin can be specifically competed off the blot (Gershoni and Palade, 1982). Also, saccharide moieties can be enzymatically modified in situ (i.e., on the blot).

B. IN SITU ENZYME MODIFICATION

The procedure is based on the fact that the glycoprotein is immobilized yet accessible to glycosidase treatment. For example, a sialoglycoprotein can be blotted and detected with wheat germ agglutinin. A replica lane can be blotted and then treated with sialidase, an enzyme that selectively removes the sialo moiety from the glycoprotein. The modification is reflected by the loss of wheat germ agglutinin binding (e.g., Gershoni and Palade, 1982; Kerjaschki et al., 1984a,b, 1986). What’s more, the underlying galactose becomes exposed, rendering the protein positive for a galactose-specific lectin such as peanut agglutinin (De Maio et al., 1986a,b). In such a way, by choosing various endo- or exoglycosidases and the right lectins, one can “sequence” an oligosaccharide side chain. Moreover, it can be asked whether a particular group is necessary for a biological function. A case in point is the fact that the binding of Sendai virus to glycophorin depends on the sialo moiety of the protein. When erythrocyte proteins are separated by SDS-PAGE, blotted, and probed with virus, a signal appears at the position of glycophorin. This signal is not obtained if the filter is treated with sialidase (Gershoni et al., 1986). For another example, there is the case of monoclonal antibodies directed against oligosaccharides, which bind differentially to glycosylated versus deglycosylated proteins. It is important to em- phasize the advantage of in situ treatment. Removal of sugar groups from proteins alters their electrophoretic mobfity. For example, the sialylated and desialylated proteins migrate differently. By treating the blot after electrophoresis, one can compare the same bands.

c. ENZYME HYDRAZIDES

A classical procedure for sugar staining is the periodic acid Schiff stain (Kasten, 1960). This method exploits the susceptibility of sugars to periodate oxidation: the vicinal hydroxyls are oxidized to generate aldehydes; these, in turn, create Schiff bases with the Schiff reagent to give a pink color. Simple application of this technique to blotted proteins is very disappointing because intolerable pink overall staining develops, obscuring any signal that may otherwise appear.

T o overcome this problem, a new reagent has been developed, namely enzyme hydrazides (Gershoni et al., 1985a). An easily detectable


enzyme (e.g., alkaline phosphatase) is modified by introducing hydrazide groups, which are highly reactive with aldehydes. The blot is quenched, treated with periodate, and subsequently probed with the enzyme-hydrazide. The latter binds to those aldehyde-containing proteins which are subsequently located by the appropriate histochemical reaction. The technique is simple and sensitive. Furthermore, it has recently been used to specifically identify surface glycoproteins (Keren et al., 1986). This is achieved by periodating intact cells. The latter are solubilized and subjected to SDS-PAGE, then blotted and probed with an enzyme-hydrazide. The fact that the periodation can be performed before electrophoresis provides the means to restrict the reaction to the proteins that are accessible on the cell surface.

3. Ligand Overlays

An extremely important application of protein blotting is in the identification and characterization of receptor proteins. The resolution of polypeptides via SDS-PAGE is a denaturing process and therefore one might assume that such treated proteins would have lost their biological activity. However, there have been many examples of the detection of enzyme or binding activity in proteins that have been SDS-denatured and resolved by PAGE (see, e.g., Lacks et al., 1979; Glenney and Weber, 1980; Hager and Burgess 1980; Lacks and Springhorn, 1980; Haggerty and Froehner, 1981; Snabes et al., 1981). It is not clear whether the activity reflects the resistance of functional domains to the denaturative effects of the detergent or the actual onset of renaturation after the gel electrophoresis.

In general, a protein preparation is subjected to SDS-PAGE, blotted, and then probed either with a substrate appropriate for a particular enzyme, a ligand corresponding to a given receptor, or a protein counterpart to a structural protein complex. Table V lists examples in which this approach has given favorable results. Notable is the work of Bennett and coworkers, who have deciphered the molecular interactions involved in the construction of cytoskeletal frameworks (Davis and Bennett, 1983, 1984; Suzuki et al., 1984; Baines and Bennett, 1985).

Various hormone receptors have been probed using ligand overlays. The ligand overlay studies of the nicotinic acetylcholine receptor has not only been quantitative, providing affinity constants of the toxin/ protein interaction, but has been the basis for identification of the toxin- binding site of the receptor (Gershoni et al., 1982, 1983; Oblas et al., 1983; Wilson et al., 1984, 1985; Neumann et al., 1985, 1986; Gershoni, 1987).


TABLE V

Ligand Overlays

figand Receptor Ref."

A d n Ankyrin (brain)

Bungarotoxin Calcium

Calmodulin

Cells Ciliary ganglionic neurons Normal rat kidney

Peripheral blood lymphocytes DNA

Epidermal growth factor dGTP Heparin Histone Human growth hormone Low-density lipoprotein

a2-macroglobulin Nerve growth factor

Pili of Gomcoccur RNA

Spectrin (brain) Streptavidin S ynapsin Transferrin

Thyroid stimulating hormone

Virus Reovirus type 3

Sendai virus Potato spindle tuber viroid

43 kDa protein ( Y I ) Torpedo membrane Spectrin fl subunit, erythrocyte anion

Acetylcholine receptor a subunit 28 kDa of bovine cerebellum and kidney Calmodulin 51 kDa phosphoprotein of rat pancreas 61 kDa protein of rat brain

Neurotrophic factors Fibronectin and 70 kDa protein

Phytohemagglutinin factor Frog virus 3 particles HeLa nuclear proteins 30 kDa cell membrane protein of human

Drosophila nuclear protein, Xenopuc ovary

Promoter-specific proteins 150 kDa membrane protein of A43 1 cells ras p21 protein apoE and apoB of human plasma HeLa nuclear proteins 67 kDa protein of liver Rabbit, bovine, human LDL receptor 160 kDa protein of adrenal cortex 125 kDa protein of human fibroblasts 130 and 100 kDa proteins of mouse

14 and 16 kDa proteins of CHO cells HeLa nuclear proteins Chloroplast 30s ribosomal protein a chain to subunit Biotin-containing proteins in plants Spectrin 90 kDa protein of NRK cells, A431 cells,

197 kDa protein of thyroid plasma

channel tubulin

(vitronectin)

neutrophils

extract

melanoma cells

and rabbit reticulocytes

membrane

67 kDa glycoprotein of rodent lymphoid

Human erythrocyte glycophorin Nuclear proteins

and neuronal cells

38 9

16,17,33 29 26

19,20 13

5 23

28 1 3 15

25

31 1 1 30 4 3 22 7 10 14 12

21 3 35 8 32

2 27

24

6

18 40


TABLE V (continwd)

Ligand Receptor Ref.“

Vinculin 215,205, and 185 kDa proteins of 39 chicken gizzard -

Zonae pellucidae proteins Spermatozoan proteins 34,36,37

Key to references: 1. Aubertin et al. (1983). 2. Baines and Bennett (1985). 3. Bowen et al. (1980). 4. Cardin et al. (1984a,b). 5. Carnow et al. (1985). 6. Co et al. (1985). 7. Daniel et al. (1983). 8. Davis and Bennett (1983). 9. Davis and Bennett (1984). 10. Dresel and Schettler (1984). 11. Fernandez-Pol(l982). 12. Fernandez-Pol et al. (1982). 13. Flanagan and Yost (1984). 14. Frey and Afting (1983). 15. Gabor and Bennett (1984). 16. Gershoni et al. (1982). 17. Gershoni et al. (1983). 18. Gershoni et al. (1986). 19. Gorelick et al. (1982). 20. Gorelick et al. (1983). 21. Gubish et al. (1982). 22. Haeuptle et al. (1983). 23. Hayman et al. (1982). 24. Islam et al. (1983). 25. Jack et al. (1983). 26. Kawasaki et al. (1985). 27. Klos et al. (1983). 28. Laurent et al. (1985). 29. Maruyama et al. (1985). 30. McGrath et al. (1984). 31. Miskimins et al. (1985). 32. Nikolau et al. (1985). 33. Oblas et al. (1983). 34. ORand et al. (1985). 35. Rozier and Mache (1984). 36. Sullivan and Bleau (1985). 37. Sullivan et al. (1983). 38. Walker et al. (1984). 39. Wilkins et al. (1983). 40. P. Wolff et al. (1985).

The spectrum of “ligands” that have been used is extremely broad. Molecules as small as single nucleotides (McGrath et al., 1984) and as large as spectrin (Davis and Bennett, 1983) have been employed. It is remarkable that blots can be probed with intact organisms. Hayman et al. (1982, 1983, 1985) showed that adherence proteins can be identified via blotting. This approach has been adopted to detect trophic factors (Carnow et al., 1985; Laurent et al., 1985). Moreover, viruses have been used to reveal their receptors on blots (Co et al., 1985; Gershoni et al., 1986).

The successful application of ligand overlay indicates that many molecular associations may rely on relatively short linear amino acid sequences or that sufficient structure can be preserved or regained during the electrophoretic chromatography and blotting. The first possibility is dependent on the intrinsic nature of the system being studied and is not amenable to experimental improvement. The second alternative will now be discussed.

A. LIMITED DENATURATION

If resolution of the constituents of a protein mixture is possible using nondenaturing gels, obviously this is advisable (see, e.g., Cohen et al., 1984). Regrettably, this is not usually the case for membrane-associated proteins, and so SDS-PAGE has been the most widely applied chromatographic procedure. However, more often than not, a high degree


of “overkill” is used. Routine protocols prescribe the boiling of a sample solubilized in 2-476 SDS + 3% 2-mercaptoethanol. The gel is then run as quickly as possible (i.e., at 30-50 mA), which in itself can generate substantial heat. Surprisingly, not many people test to see the separation obtained under much milder conditions-for example, by not heating the sample, by omitting reducing agents, or by decreasing the detergent concentration. Many samples can be effectively solubilized in 0.5% SDS or less. Running the gel in the cold is also advantageous. For this purpose, Delepelaire and Chua (1979) developed lithium dodecylsulfate/polyacrylamide gels. Such gels have been employed in the protein blot analysis of the nicotinic acetylcholine receptor (Gershoni et al., 1982, 1983). Unexpected factors can also have detrimental effects. Bromphenol blue can sometimes act as a cross-linker and alkylate some proteins. Therefore one should attempt to obtain reasonable resolution with minimal modification of the proteins.

B. RENATURATION

The subject of protein refolding has been reviewed extensively (see, e.g., Wetlaufer and Ristow, 1973; Anfinsen and Scheraga, 1975; Wet- laufer, 1981; Kim and Baldwin, 1982). However, no simple formula exists, especially for membrane-associated proteins. A number of protocols have been suggested, yet none have been rigorously examined and controlled. After SDS-PAGE, one usually wants to remove excess SDS and allow the protein to refold in situ. This of course may create difficulties for elution of the protein out of the gel, since the depletion of SDS can be accompanied by loss of net negative charge. Therefore, Bowen et al. (1980) applied diffusion blotting, which is charge inde- pendent. Alternatively, one can transfer under renaturing conditions, for example, by replacing the SDS with more favorable nonionic or zwitterionic detergents (Mandrel1 and Zollinger, 1984) or urea (Frey and Afting, 1983). Regardless what approach is taken, most probably each case will have to be worked out empirically.

4. Nucleic Acid Overlays

Probing protein blots with nucleic acids is a special case of ligand overlays. This is not really any different from the examples described above but, rather, the interactions discovered are of special interest. The regulation of gene expression is assumed to rely on the selective recognition of specific proteins with discrete structures/sequences in nucleic acids. Therefore, protein blots have been prepared and probed with DNA and RNA (Bowen et al., 1980; Aubertin et al., 1983; Gabor and Bennett, 1984; Rozier and Mache, 1984).


The work of P. Wolff et al. (1985) is noteworthy because the detection process entailed a secondary nucleic acid probe. Nuclear proteins were resolved on SDS gels and blotted to NC. The blots were probed first with potato spindle tuber viroids and then after fixation reprobed with ”P-labeled complementary DNA.

5. Additional Applications

Protein blotting can provide a means to attain certain objectives besides the detection of molecules via overlay techniques. As has already been indicated, blots have been used for the purpose of isolation of monospecific antibodies for subsequent immunocytochemistry (Olmsted, 198 1 ; see Section XIII. 1 .A.a). The blotted material can also be used as an antigen. Proteins are separated on SDS gels and blotted; a certain band is identified (e.g., by Amido black staining), and this band is excised from the blot. So in essence the blotting has been used for the isolation of a specific polypeptide. By blotting to glass fiber filters, such isolated polypeptides can then be subjected to microsequencing (Van- dekerckhove et al., 1985; Aebersold et al., 1986). Alternatively, isolated polypeptides can be used for immunization. A number of possibilities exist. The filter can be chopped up and introduced subcutaneously or intraperitoneally. However more efficient presentation of the antigen to the immune system can be achieved by eluting the protein off the immobilizing matrix (P. J. Anderson, 1985; Knudsen, 1985; Parekh et al., 1985). Two general approaches have evolved. One is by leaving the matrix intact and eluting off the bound antigen into a solution to be later injected. Elution can be achieved by the use of nonionic detergents; dimethyl sulfoxide has also been found useful for this. The second approach requires disruption of the matrix. For example, NC is dissolved in acetone or high concentrations (> 90%) of methanol. Thus a polypeptide-containing piece of NC is dissolved and the resultant slurry is either extracted to obtain the protein in an aqueous phase or simply mixed with adjuvant and used directly. Clearly a “smart membrane” would be useful. Such a membrane would bind protein via a reversible cross-linking moiety. But alas, as long as blotting matrices are in essence “misused” filtration media, we blottologists will simply have to make do.

One application that actually exploits the filtration characteristics of immobilizing matrices is the concentration of dilute factors secreted by cells grown in culture (Luscher and Gitler, personal communication). Cells are cultured in serum-free medium. Large volumes of “spent” medium are then filtered through a small disc of NC [for this application


a large pore size of 0.8 pm (AE 91, Schleicher and Schuell, Inc.) is advantageous to prevent clogging]. The disc adsorbs the very dilute protein and in this way concentrates the contents of even 5 ml onto the surface of a 50 mm2 disc. The disc can then be inserted into the well of a slab gel, the protein eluted off, and in this manner directly analyzed by SDS-PAGE. The elution of the bound protein requires a nonionic detergent because SDS by itself is insufficient.

The in situ enzymatic modification of blotted polypeptides has already been discussed for glycoproteins (see Section XIII.2.B). Valtorta et al. (1986) have also employed such an approach in the assay of the phosphorylation of blotted proteins. Moreover, the phosphoproteins can be further analyzed by in situ proteolysis followed by phosphopep- tide mapping.

Protein blots can be used to allow multiple probings of one sample. Diffusion blotting, by nature, generates two replicas of a gel. Electrob- lotting can generate many replicas by either blotting one gel a number of times and each time changing the filter (Legocki and Verma, 1981; McLellan and Ramshaw, 1981; Manabe et al., 1985) or by placing a series of filters one behind the other and blotting exhaustively in the absence of methanol (Gershoni and Palade, 1982). Both procedures, however, produce replicas of varied quality. The first and last replica undoubtedly are different; therefore caution should be taken. Another possibility is to probe a blot more than once. If each probe is distinctive in its own right, a blot can be double-labeled or probed sequentially without special treatment (Neumann et al., 1985). Alternatively, the first signal can be removed prior to the reprobing. This is possible by treating the blot in low-pH buffer, SDS, or urea and 2-mercaptoethanol (Legocki and Verma, 1981; Reiser and Wardale, 1981; Symington et al., 1981; N. L. Anderson et al., 1982; Erickson et al., 1982; Gullick and Lindstrom, 1982).

XIV. SUGGESTED PROTOCOLS

This section provides some of the “nitty-gritty” of selected protocols. These lists are to be regarded as reasonable starting points to be tailored to the reader’s particular needs.

1. Dot-Blot Protocol

(i) Samph application. Apply an aliquot of a protein mixture te squares (0.9 X 0.9 cm) of immobilizing matrices. The application can be


performed using a micropipette; 1-2 pl is fine and no more than 5 pl at once should be attempted. A Beckman single-point sample applicator (no. 324399) can also be used. The use of dot-blot manifolds, in which larger volumes can be applied, has been discussed in Section IV.2. To test the suitability of different matrices, a set of dot blots can be prepared for each type. The matrix can be dry so that the droplet is quickly absorbed. The squares are then wetted in an appropriate buffer, for example, Tris-HC1, pH 7.4, or phosphate-buffered saline (PBS). Wetting the filters should be accomplished by floating them onto the surface of the buffer; wetting both sides simultaneously by sub- merging the filters may entrap air bubbles in the depth of the filter. The number of squares to be used should accommodate the number of treatments or variables being tested. Each filter should be dealt with individually. Separate filters can be conveniently treated using a 24-well tissue culture dish.

(ii) Quenching. One should test various concentrations and types of proteins or nonionic detergents as quenchers. Time and temperature of quenching should also be considered. At least one square should be used to control the absence of quench. As illustrated in Fig. 1, quenching is not always necessary (see also Cardin et al., 1984a,b).

(iii) Probing. Here too the optimal concentration of the probe, as well as time and temperature of incubation, should be determined. At least one filter should be devoted to the “minus probe” control when a second antibody is to be used. The presence of ions, or specific buffer conditions that may optimize binding, should also be tested.

(iv) Wmhing. The removal of excess unbound probe is achieved by washing (aspiration of the incubation fluid replaced by new buffer). In dot-blot assays the washing is often not very efficient, due to the relatively small volume of wash solution used (-1 mY filter in a 24-well plate). In all cases, good vigorous shaking is advisable-something resembling the rhythm of an Israeli hora. Washes should consist of frequent changes of the wash solution. The presence of quenchers does not usually add to the wash efficiency. On the other hand, nonionic detergents usually reduce the extent of background. Such reagents should be used with caution, however, because they can reduce the very signal one wishes to obtain (see Section IX). The minimal adequate wash should be determined. Excessive washing may lead to loss of


signal, especially when the protein-ligand complex is of low avidity. Washing with ice-cold buffer may help in this respect.

(v) S e c d probe. If a second probe is necessary, it should be calibrated in the same manner as described in step iii.

(vi) Final wash. Repeat step iv. (vii) Detection of signal. To evaluate the success of one protocol as

compared to that of another, detection of the complex is necessary. When an enzyme-linked procedure is used, the filters are incubated in a substrate solution and the results are visible. One can test different substrates and different reaction conditions. If this in itself is the sole object of the dot blot, steps i-vi are not necessary and direct application of the enzyme conjugate to the filter is sufficient. If radioactive probes are used, one should autoradi- ograph the filters. Granted, counting the filters may appear to be a more convenient and quantitative way to monitor the results, yet this often proves to be misleading. The signal one wishes to obtain should be a well-defined dense dot on a clear background. The absolute amount of radioactivity specifically bound to the spot may be merely a few hundred counts above a substantial amount (a few thousand counts) of evenly diffused nonspecific background. Counting the filters in such an instance would be disappointing, whereas the appropriately exposed autoradiogram could be quite pleasing. Counting the filters after autoradiography is then always possible.

4. Electroblotting

A. PREPARATION

(i) Prepare cold (4°C) transfer buffer, 7.6 g Tris + 36 g glycine in 4

(ii) Cut NC filter slightly larger than the area of the gel to be

(iii) Run the SDS-polyacrylamide gel of your choice.

liters of water (a 10-fold concentrate is easily made).

transferred.

B. BLOTTING

(i) Add transfer buffer to the blotting apparatus, which already contains the cassette with porous pads (Scotch-Brite). In this way the pads are well wetted. Pour a bit of excess buffer into a separate tray or box in which to wet the NC filter (wet by flotation; see


Section XIV.l). Reuse of transfer buffer may be possible (Good- erham, 1984).

(ii) Take out the cassette and pads and lay them horizontally on a tray.

(iii) Open up the cassette so that the mechanical support and one pad are facing up; set aside the excess pads and the second half of the cassette. If individual lanes are to be blotted separately, cut the gel accordingly. In all cases the stacking gel should be removed and not blotted, otherwise a gooey residue adheres to the blot and interferes with subsequent probing.

(iv) Place the gel(s) directly onto the pad, make sure the orientation (lefdright, tophottom) of the gel is clear. When a gradient electric field is used, be certain that the top of the gel is placed in the area of the maximal field.

(v) Wet the surface of the gel with transfer buffer and apply NC. It is important that air bubbles not be trapped between the gel and the NC.

(vi) Place a second pad onto the gel-NC sandwich. Make an additional assembly if more than one gel is to be transferred. Otherwise add pads as required to ensure snug packing and support of the gel and filter, and close the cassette with its second frame.

(vii) Insert the cassette into the blotting apparatus and transfer for at least 2 h at about 5-8 V/cm (30-50 V, -200 mA). When blotting SDS gels, the filter is oriented to face the positive electrode.

(viii) After transfer, remove the filter from the cassette. The gel can be stained to evaluate the efficiency of transfer. The filter can be stored or used directly.

3. Storage of Blots

The ability to store a blot before use is very much dependent on the individual system being studied. It has been reported that blots can be air-dried and stored or up to 6 months (N. L. Anderson et al., 1982; Douglas and King, 1984). On the other hand, some antigenic deter- minants or receptor binding sites will not tolerate being dehydrated, so drying the blot would be unacceptable. The analysis of glyco moieties should be completely indifferent to drying the blot. If drying is permissible, placing the blot between two sheets of Whatman 3MM paper is adequate. Such filters have been stored in a drawer and probed with concanavalin A 2 years later and found to give the same results


obtained when the blot was fresh (Gershoni and Palade, 1982, and unpublished results). When drying is detrimental, a blot should be used immediately or kept in a buffered solution for as long as is empirically permissible. Freezing damp blots placed between plastic wrap is sometimes quite acceptable for extended storage.

4. Processing Blots (i) Quenchzng. When NC is used, quenching for 1-2 h at room

temperature is usually sufficient in 0.5% hemoglobin or 1-3% BSA in PBS (or Tris/NaCl buffer); both serve as an efficient average quench. Other quenchers that seem to work well are 10% fetal calf serum or 10% low-fat milk. Milk and hemoglobin seem to be the best quenchers for PCM.

(ii) Probing. For immunoblotting, the quenched blot is incubated in serum diluted in a buffer, such as PBS, containing quencher. For whole rabbit serum an initial dilution of 1 :200 is reasonable. Monoclonal antibodies usually have to be used at much higher concentrations, so medium should be tested at 1 : 10 dilution. Obviously, purified immunoglobulins or ascites fluids must be diluted appropriately (about 10 pg IgC/ml should be sufficient). Most other probes are diluted into a quenching solution according to their radioactivity or micrograms-per-milliliter value. Generally, lo5 cpm per milliliter of solution is enough and no more than lo6 cpdml is normally required. Nonradioactive probes can be used at about 10 pg/ml. An hour or two of probing is most often enough.

(iii) Washing. A number of washes in buffer without quencher is usually sufficient-for example, four washes of 10 min in a large volume of buffer (25-100 ml depending on the size of the filter and container). A detergent wash can help decrease background (0.1% Tween 20, 5-10 min).

(iv) Second Probes. When a second probe is used (e.g., radioiodinated protein A) the washed filter is simply reprobed as described above followed by a wash cycle.

5. Detection of signal A. AUTORADIOGRAPHY

When autoradiography is used, the washed blots should be kept damp and placed between plastic wrap as described in Section XI. 1. Keeping the blot damp will allow subsequent reprobing or further manipulation (drying the blot often irreversibly denatures the probe to the surface of the filter such that it can no longer be removed).


B. DETECTION OF ENZYME-CONJUGATED PROBES

Alkaline phosphatare conjugates are detected by the incubation of the blot in naphthol AS-MX-phosphate (free acid, 10 mg) dissolved in 200 pl of dimethylformamide, which is then added to 100 mM Tris-HC1, pH 8.4 (100 ml), and 30 mg of fast red TR salt. Within 15-30 min a red precipitate is formed at the position of the enzyme reaction.

Honerd i sh peroxidme is detected using 40 mg of diaminobenzidine (tetrachloride salt) dissolved in 100 ml of PBS to which 70 pl of H202 (from a 30% stock solution) is added. The substrate is to be prepared fresh and stored in the dark until use. The brown precipitate appears almost instantaneously and is light sensitive. An alternative substrate for horseradish peroxidase is 4-chloronaphthol (10 mg) instead of the diaminobenzidine. This will give a dark blue precipitate. Intensification of signals can be obtained by introducing ions such as cobalt or nickel to the reaction solutions (De Blas and Cherwinski, 1983).

c. GENERAL PROTEIN STAINING OF BLOTS

The general protein pattern on the blot can be easily demonstrated by staining the blot. A few procedures are listed.

(i) Incubate the blot in 0.1% Amido black in 25% isopropanoYlO% acetic acid and destain in the same solution minus the dye (Gershoni and Palade, 1982).

(ii) A Ponceau S solution (0.1%) in 7% trichloroacetic acid in water may be used. Destaining in water gives a transient pink patter.i. A permanent pattern is obtained by destaining in 25% isopropanoY 10% acetic acid.

(iii) Negative staining of the blot is also possible. This is achieved by incubating the blot in a dilute solution of alkaline phosphatase (10 pg/ml). The blot is then rinsed in PBS and reacted for enzyme activity as described above.

XV. TROUBLESHOOTING

In a multistep process such as immunoblotting there are numerous points that may be problematic. In this section, I deal with three problem areas and provide possible explanations that sometimes help in overcoming them.


1. Transfer Technique

The quality of the blot will be directly affected by the mode of transfer used. Some of the problems in transfer technique are listed below.

A. BIASED TRANSFER

High molecular weight polypeptides are eluted out of the gel much more slowly than are low molecular weight proteins. The result is misrepresentation of the original protein pattern. Solutions to this problem are the use of gradient gels in combination with a gradient electric field, longer transfer time, and a decrease in the ionic strength of the buffers. The latter allows the increase of the potential difference without generating excessively high currents. It should be emphasized that prolonged blotting times may elute the high molecular weight proteins yet drive the low molecular weight proteins through the matrix. This situation can be reduced by using matrices of smaller pore size or by using charge-modified nylon. Other approaches are to use reversible cross-linkers in the acrylamide polymer (Renart et a]., 1979; Tas et al., 1979; Bolen et al., 1982) or proteolytic nicking of high molecular weight proteins while electroblotting (Gibson, 198 1).

B. STUBBORN BANDS

Periodically one runs into a “stubborn band”-a polypeptide that for some reason does not elute even though it is of moderate molecular weight. This polypeptide may be at its isoelectric point due to the removal of SDS during the blotting. Use of a more alkaline buffer can be helpful. This approach has been used in particular for the blotting of relatively basic proteins (e.g., Szewczyk and Kozloff, 1985).

c. BALDSPOTS

Staining a blot sometimes reveals not only the protein pattern but bald spots, areas where transfer did not occur. Such blemishes should and usually can be avoided. The most common reason for bald spots is air bubbles caught between the gel and the filter during transfer. Care should be taken when applying the filter to the gel to expel all air bubbles. It also appears that the use of blotting paper (e.g., Whatman 3MM) as a protective barrier between the gel and Scotch Brite pad or between the immobilizing matrix and Scotch Brite pad can cause problems. Whereas this practice is intended to provide more gentle care for the blot, it is sometimes detrimental because air can be trapped


by the paper as well. Another possible cause for bald spots is the use of old NC, which may not wet uniformly.

D. DISTORTED BANDS

The geometry of a transferred band is sometimes distorted. This may be the result of insufficient contact between the gel and filter. In assembling the gel-filter sandwich with the pads, it is important to ensure a snug fit. Loosely held gels tend to give slurred or skewed bands.

2. Signal Quality

The frustration resulting from the development of “blank” autoradi- ograms should be experienced as seldom as possible. Obviously, there are times when the lack of signal actually reflects a true result. Sometimes, however, signals of relevance can be generated by modification of methods rather than by changing biological samples or probes. Optimization of binding conditions is a good first step. This can be done by dot blotting to check the specific requirements for your particular system (e.g., pH, salt concentration, divalent cations). Once the best conditions have been established, treatment of the sample should be considered. Limited denaturation of the sample can help (see Section XIII.3.A); postblot renaturation may be feasible. The incubation of the blot with the probe can be optimized; longer incubation times with higher probe concentrations may be necessary. It should be remembered that the intact receptor, for example, may bind its ligand a number of orders of magnitude more efficiently than does the dissociated individual subunit. Washing the blot in cold buffer may preserve labile complexes.

In general, if a dot blot gives a negative result, the chances are slim that a signal will be obtained on a gel transfer. The rule of thumb is that longer incubation time and increased probe concentrations may help; however, they usually lead to higher background rather than specific signal.

3. Background Problems

Intolerable background is a problem that can sometimes be resolved. The important first step is to generate a uniform, rather than a blotchy, background.

A. UNEVENBACKGROUND

There are a number of reasons for uneven nonspecific background. The incubation conditions should be suspected first. “Seal-a-meal” bags


are routinely employed in nucleic acid blotting and are extremely useful; however this practice is not as beneficial for protein blotting. The quenching, incubations, and washes should be performed in as large a volume as possible so that the filter is freely bathed in the different solutions and does not get stuck to the sides of the container. Shaking or rocking the filters is necessary to ensure constant good contact with the solutions. Excessive shaking in a container too small for a blot sometimes leads to bald spots. This is due to the formation of a “node” in the rocking solution where fluid does not efficiently mix.

Blotches and spots can develop from uneven wetting of the blot, the use of old filters that do not wet uniformly, or the aggregation/ precipitation of quenchers (this is especially true for hemoglobin). Quencher precipitation can be reduced by quenching at room temperature, filtration of the quencher solution, and thorough washes between incubations.

Often, once uniformity of a background, albeit high, has been achieved, a presentable signal can still be obtained by regulating the exposure time of the autoradiogram (or incubation time with the enzyme substrate). Although this is one approach, efforts should be made to reduce background as much as possible.

B. HIGH BACKGROUND

Optimizing the quench should be done by dot blotting. When excessive background is generated, one should consider changing one or more of several possible parameters (including the quench solution itself), increasing the quencher concentration, quenching at higher temperature, andor introducing a detergent (e.g., Tween 20) into the washing regimen. Most often NC filters give clearer background than do PCM; however the opposite is true in some instances. The use of milk as a quencher for PCM seems quite favorable.

c. FALSE POSITIVES

I have no good suggestions with regard to false positives. In some cases a probe binds nonspecifically to the entire protein pattern, generating signals for every band. Curiously, in certain instances the probe can distinguish between SDS-denatured protein and native protein. For example, when BSA is used as a quench, only the SDS-electrophoresed BSA in the protein standard is labeled, not the overall background. This may indicate that the interactions are hydrophobic; the SDS- denatured protein exposes hydrophobic domains that are otherwise well hidden in the nondenatured protein. Indeed, sometimes detergent


washes tend to reduce false positives. Goat antibodies tend to be more “sticky” than rabbit antibodies, and S. aureus protein A seems to cause even fewer false positives. Therefore, when possible one should try different second probes.

XVI. CONCLUDING REMARKS

It has been my intention to systematically analyze each step of a blotting experiment in the hope that the reader will gain insight, a true feeling for what is known on this subject. By using this information to modify existing protocols and thus “fine-tune” them to one’s specific needs, optimal results should be more easily achieved. Clearly, there is much that is still insufficiently understood. Hopefully, as more studies are conducted, the obstacles in applying blot techniques will be overcome and in this way more biological questions may be closer to their answer.

ACKNOWLEDGMENTS

I thank the many colleagues who have helped me in preparing this manuscript. Especially important have been Nigel Cox and Fred E. Davis of the Yale University School of Medicine Medical Instrument Facilities, who not only encouraged me in pursuing more efficient blot apparatus but gave essential help in preparing all the prototypes of the apparatus described in Figures 2 and 3; Dr. George E. Palade, who has supported me and my work from my very first blot; the Weizmann Institute of Science workshop for preparing the revised version of the blot apparatus used in my laboratory; Rachel Samuel for typing the manuscript; Dvorah Ochert for her comments and assistance in editing it; Edward A. Bayer, Charles L. Jaffe, Antonio De Maio, and Ann Chayen for their constructive criticism; Steve Karlish for a comment; and Jean-Michel Lebleautte for preparing Figures 4 and 5 and all the tables.

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Protein Blotting: A Manual

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