NOTES & TIPS 263

BSA as the standard, a value that agreed extremelywell with that obtained above (0.014 mg/ml).

In sum, the ability to assay the specific activities ofenzymes or transporters in reconstituted vesicles re-quires accurate determinations of protein content. Asstandard protein determination methods are often in-sufficient when low amounts of protein are used orwhen reactive compounds are present, a revised mem-brane protein quantitation assay was required. To thisend, the amido black protocol presented here is idealfor studies using reconstituted or native membranevesicles.

Acknowledgments. The preparation of vacuoles by Sean Reid anddiscussions with Bob Duda on protein determination methods weredeeply appreciated. This work was supported by Grant MCB-9506002 from the National Science Foundation.

REFERENCES

1. Brodsky, J. L., Hamamoto, S., Feldheim, D., and Schekman, R.(1993) J. Cell Biol. 120, 95–102.

2. Brodsky, J. L., and Schekman, R. (1993) J. Cell Biol. 123, 1355–FIG. 1. (A) Scanned image of an amido black-stained blot. Dupli- 1363.cate samples of BSA and single samples of ER- and vacuole-derived

3. Roberts, C. J., Raymond, C. K., Yamashiro, C. T., and Stevens,reconstituted vesicles were analyzed as described in the text. (B)T. H. (1991) Methods Enzymol. 194, 644–659.Standard curve obtained from a spectrophotometric analysis of the

4. Rexach, M. F., Latterich, M., and Schekman, R. (1994) J. CellBSA samples in A (r Å 0.996).Biol. 126, 1133–1148.

5. Minamide, L. S., and Bamburg, J. R. (1990) Anal. Biochem. 190,66–70.

tively. Reactions containing only the reconstitution6. Zlatanova, J., Paneva, E., and Yaneva, J. (1994) Cytobios 78,

buffer or up to 12 mg of lipid failed to yield a positive 135–138.result in this assay (see Fig. 1A). 7. Wessel, D., and Flugge, U. I. (1984) Anal. Biochem. 138, 141–

To determine if the final concentration of protein re- 143.flected polypeptides that had been inserted into theliposomes, reconstituted vesicles prepared from ERmembranes were floated in a sucrose gradient as pre-viously described (1). During centrifugation, unincor-porated proteins remain in the bottom of the centrifuge Removal of Chromosomal DNA by Mg2/

tube while reconstituted proteins ascend through the in the Lysis Buffer: An Improved Lysisgradient (1). The floated band of ER reconstituted vesi- Protocol for Preparing Escherichia colicles was removed for an amido black protein determi-

Whole-Cell Lysates for Sodium Dodecylnation and a protein concentration of 0.014 ({0.002)Sulfate–Polyacrylamide Gel Electrophoresismg/ml was obtained. This value is about one-half of

that obtained using the unfloated vesicles (0.030 mg/ml, see above), suggesting that only a fraction of total Min Chen and Philipp Christenprotein incorporates into the liposomes, as observed

Biochemisches Institut der Universitat Zurich, Switzerlandpreviously (1).Finally, to demonstrate that the technique described

Received November 26, 1996here is valid, an alternate, more time-consuming pro-tein determination method was developed. First, deter-

Analysis of Escherichia coli whole-cell lysates bygent and lipids from ER-derived floated reconstitutedvesicles described above were chloroform–methanol SDS–PAGE combined with either silver staining or

Western blotting is a frequently used test for the ex-extracted using the method of Wessel and Flugge (7).The final precipitated protein pellet was dissolved in pression of target proteins. A problem associated with

whole-cell lysates is their high viscosity caused by chro-100 ml of 1% SDS and the concentration of protein wasdetermined using the BCA kit from Pierce (Rockford, mosomal DNA which renders the lysates unpipettable.

Sonication and repeated passage through a hypodermicIL). Using this analysis, the protein concentration ofthe floated material was assayed at 0.013 mg/ml with needle have been proposed for fragmentating chromo-

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NOTES & TIPS264

DNA, it can cause the formation of breakdown productsof some proteins (Fig. 1). Compared with sonication orrepeated passage through a hypodermic needle, theMg/SDS lysis procedure is more efficient in reducingviscosity and much simpler to perform.

The presence of 0.1 M magnesium chloride in thesamples does not alter the electrophoretic behavior of

FIG. 1. Western blot of E. coli whole-cell lysates prepared with the proteins. To avoid the preparation and storage ofdifferent protocols. E. coli cells (1 ml of saturating culture) harboring two different sorts of sample buffers, the Mg/SDS lysisa mutant of aspartate aminotransferase (AspAT) were lysed with

buffer can be used in place of Laemmli sample buffer100 ml Mg/SDS lysis buffer or 100 ml Laemmli sample buffer. Samples(3) for soluble protein samples as well.of 5 ml were fractionated with SDS–PAGE (10%) and electrotrans-

ferred to a nitrocellulose membrane. The blot was developed with In short, cell lysates devoid of chromosomal DNA andantiAspAT antiserum, an alkaline phosphatase-labeled second anti- free of heating artifacts can be obtained with extremebody, and 5-bromo-4-chloro-3-indolyl phosphate (Sigma) plus nitro- ease with the Mg/SDS lysis procedure.tetrazolium blue chloride (Fluka). Lane 1, sample prepared with Mg/SDS lysis buffer and incubated 2 min at 1007C. Lane 2, sample pre-pared with Mg/SDS lysis buffer without heating. Lane 3, sample REFERENCESprepared with Laemmli sample buffer and incubated 2 min at 1007C

1. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular(the sample prepared with Laemmli sample buffer without heatingCloning. A Laboratory Manual, Cold Spring Harbor Laboratorywas unpipettable). The arrow indicates a degradation band in thePress, Cold Spring Harbor, NY.heated samples.

2. Neidhardt, F. C., Ingraham, J. L., and Schaechter, M. (1990)Physiology of the Bacterial Cell. A Molecular Approach, Sinauer,Sunderland, MA.

somal DNA (1). DNase has also been used for the same 3. Laemmli, U. K. (1970) Nature 227, 680–685.purpose. Alternatively, exposure of the jelly-like SDSlysates to 1007C for 1 to 2 min will precipitate the DNAand thus reduce the viscosity. However, heating maycause the formation of breakdown product of target

A Mammalian Gene Expression Vector withproteins. Here we describe a new method with whichchromosomal DNA is easily removed without heating. Blue–White Selection for Efficient Subcloning

In E. coli cells, the chromosomal DNA is in a compact in Escherichia coliform called nucleoid. When the cells lyse, their DNAspills out into long strands making the medium highly

Zhong Liu,* Linda M. Cashion,* Li Zhu,†viscous. However, it was observed that the nucleoidsand Ying Luo†,1

remained condensed and the lysate was not viscous*Berlex Biosciences, 15049 San Pablo Avenue, P.O. Boxwhen the cells were lysed by mechanic means in the4099, Richmond, California 94804; and †CLONTECHpresence of Mg2/ in high concentration (§1 M) (2). WeLaboratories, Inc., 1020 East Meadow Circle,therefore explored the feasibility of including MgCl2 inPalo Alto, California 94303-4230SDS lysis buffer for preparing whole-cell lysates and

found the following composition of lysis buffer mostReceived November 1, 1996effective in lysing the cells and precipitating chromo-

somal DNA, as well as denaturing proteins and dissoci-ating protein complexes: Gene cloning is one of the essential steps in the study

of protein structure and function. The pUC series ofMg/SDS lysis buffer. Buffer consists of 0.1 Mplasmids are the most widely used for this purposeMgCl2, 4% SDS, 10% glycerol, 5% 2-mercaptoethanol,because of their reliable and convenient blue–white0.01% bromophenol blue, and 100 mM Tris chloride, pHselection feature in Escherichia coli (1). These types of6.8.vectors, however, have limited value in mammalianMg/SDS lysis procedure. (i) Spin 1 ml of a satu-cells due to the lack of a mammalian transcription unit.rated E. coli culture (about 109 cells per milliliter) in aWith rapid progress of the human genome project, hun-microfuge for 45 s. (ii) Add 100 ml of Mg/SDS lysis bufferdreds of thousands of new cDNA sequences have beento the cell pellet and vortex for 1 min to resuspend andcloned (2). A significant portion of these cDNA frag-lyse the cells. (50 ml or less Mg/SDS lysis buffer mayments will be transferred to eukaryotic expression vec-be used to obtain more concentrated lysates for thetors for subsequent structural and functional analysis.detection of low-expression target proteins). (iii) Spin

2 min in a microfuge to remove the precipitate. Theclear supernatant can now be used for SDS–PAGE. 1 To whom correspondence should be addressed. Fax: 415-354-

0776.Although heating is equally effective in removing

ANALYTICAL BIOCHEMISTRY 246, 264–267 (1997)ARTICLE NO. AB972033

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