Electrophoresis and Blotting Techniques

Asheesh Pandey, IET Lucknow1

Electrophoresis

2

Electrophoresis is a method whereby charged molecules in solution, mainly proteins and nucleic acids, migrate in response to an electrical field.

Their rate of migration through the electrical field, depends on:

- the strength of the field, - on the net charge, size, - and shape of the molecules, - and also on the ionic strength, viscosity, and temperature of the medium in which the

molecules are moving.

As an analytical tool, electrophoresis is simple, rapid and highly sensitive.

It can be used analytically to study the properties of a single charged species or mixtures of molecules. It can also be used preoperatively as a separating technique

Asheesh Pandey

Electrophoresis

3

Electrophoresis is usually done with gels formed in tubes, slabs, or on a flat bed.

In many electrophoresis units, the gel is mounted between two buffer chambers containing separate electrodes, so that the only electrical connection between the two chambers is through the gel.

Separation according to migration of charged particles in electric field Electrolysis: Chemical decomposition supplemented by current (components reach electrode) F = v f = q E

V=Velocity, E=Electric field, q=Charge f=Frictional coefficient

Asheesh Pandey

Electrophoretic mobility

4

Electrophoretic mobility μ = v / E = q / fV=Velocity, E=Electric field, q=Charge f=Frictional

coefficientDepends on

Particle property :Surface, charge, density & sizeSolution properties: Ionic strength, pH,

Conductivity,viscosityTemperature & Voltage

Asheesh Pandey

In most electrophoresis units, the gel is mounted between two buffer chambers containing separate electrodes so that the only electrical connection between the two chambers is through the gel.

5 Asheesh Pandey

The Technique

6 Asheesh Pandey

The Technique

7 Asheesh Pandey

Slab Gel Unit

8 Asheesh Pandey

Flat Bed Unit

9 Asheesh Pandey

Interrelation of Resistance, Voltage, Current and Power

10

Two basic electrical equations are important in electrophoresis

The first is Ohm's Law, I = E/R

The second is P = EI

This can also be expressed as P = I2R

In electrophoresis, one electrical parameter, either current, voltage, or power, is always held constant

Asheesh Pandey

Consequences

11

Under constant current conditions (velocity is directly proportional to current), the velocity of the molecules is maintained, but heat is generated.

Under constant voltage conditions, the velocity slows, but no additional heat is generated during the course of the run

Under constant power conditions, the velocity slows but heating is kept constant

Asheesh Pandey

The Net Charge is Determined by the pH of the Medium

12

Proteins are amphoteric compounds, that is, they contain both acidic and basic residues

Each protein has its own characteristic charge properties depending on the number and kinds of amino acids carrying amino or carboxyl groups

Nucleic acids, unlike proteins, are not amphoteric. They remain negative at any pH used for electrophoresis

Asheesh Pandey

Temperature and Electrophoresis

13

Important at every stage of electrophoresisDuring Polymerization

Exothermic ReactionGel irregularitiesPore size

During ElectrophoresisDenaturation of proteinsSmile effectTemperature Regulation of Buffers

Asheesh Pandey

What is the Role of the Solid Support Matrix?

14

It inhibits convection and diffusion, which would otherwise impede separation of molecules

It allows a permanent record of results through staining after run

It can provide additional separation through molecular sieving

Asheesh Pandey

Agarose and Polyacrylamide

15

Although agarose and polyacrylamide differ greatly in their physical and chemical structures, they both make porous gels.

A porous gel acts as a sieve by retarding or, in some cases, by completely obstructing the movement of macromolecules while allowing smaller molecules to migrate freely.

By preparing a gel with a restrictive pore size, the operator can take advantage of molecular size differences among proteins

Asheesh Pandey

Agarose and Polyacrylamide

16

Because the pores of an agarose gel are large, agarose is used to separate macromolecules such as nucleic acids, large proteins and protein complexes

Polyacrylamide, which makes a small pore gel, is used to separate most proteins and small oligonucleotides.

Both are relatively electrically neutral

Asheesh Pandey

Agarose Gels

17

Agarose is a highly purified uncharged polysaccharide derived from agar

Agarose dissolves when added to boiling liquid. It remains in a liquid state until the temperature is lowered to about 40° C at which point it gels

The pore size may be predetermined by adjusting the concentration of agarose in the gel

Agarose gels are fragile, however. They are actually hydrocolloids, and they are held together by the formation of weak hydrogen and hydrophobic bonds

Asheesh Pandey

Structure of the Repeating Unit of Agarose, 3,6-anhydro-L-galactose

18

Basic disaccharide repeating units of agarose,

G: 1,3-β-d-galactose

and

A: 1,4-α-l-3,6-anhydrogalactose

Asheesh Pandey

Gel Structure of Agarose

19 Asheesh Pandey

Polyacrylamide Gels

20

Polyacrylamide gels are tougher than agarose gels

Acrylamide monomers polymerize into long chains that are covalently linked by a crosslinker

Polyacrylamide is chemically complex, as is the production and use of the gel

Asheesh Pandey

Crosslinking Acrylamide Chains

21 Asheesh Pandey

Components of Lysis Buffer

22

Buffer (Tris or Hepes buffer with pH 7-8)Salt (usually NaCl 150mM (low) to 500mM(high)Chelating agent (EDTA or EGTA)DetergentProtease inhibitorPhosphatase inhibitor (optional)

Asheesh Pandey

Lysing Cells

23

Treat cells with appropriate conditions depending on the experiment

Pellet cells and lyse them in the appropriate lysis buffer.

Most important ingredient in lysis buffer is detergent. Most stringent to weakest

SDSNP-40Triton X-100Tween 20DigitoninCHAPSBrig

Asheesh Pandey

24

Second most important ingredient is protease inhibitors.Once proteins are denatured by detergents, they are

susceptible to degradation by proteases.Need more than one inhibitor since there are lots of

proteases.Protease inhibitors

AprotininLeupeptinPMSF (phenylmethylsulfonyl fluoride)

Add immediately before lysis since PMSF activity decreases over time in aqueous solutions. About 15-30 minutes of activity.

Asheesh Pandey

Phosphatase inhibitors

25

Need to add to lysis buffer if using phopshospecific antibodies or suspect protein of interest is phosphorylated

InhibitorsZnCl2

NaFNa-Vanadate (tyrosine phosphatase inhibitor, add prior

to lysis since only active in pH 7 for minutes)

Asheesh Pandey

Further denature proteins

26

Add lysate either a known concentration of proteins or cell number equivalent to SDS loading buffer

SDS Loading BufferBuffer (Tris-Cl pH 6.0)2% SDS0.1% bromophenol blue10% glycerol (allows sol’n to sink to bottom of gel wells)-mercaptoethanol ( reducing agent)

SDS loading gel mixed with lysate is boiled to further denature proteins. (104gm SDS/1gm Protein)

1:10 ratio loading buffer to lysateAsheesh Pandey

Ingredients in Gel

27

Sodium dodecyl sulfate (SDS)Tris buffer (either glycine or tricine)Acrylamide and NN-bis-acrylamide

Forms gel matrixTEMED

Catalyst for polymerization (produces free radials from APS)Ammonium persulfate (APS)

Source of free radials for polymerization

Could purchase pre-cast gels if you have the money.

Asheesh Pandey

Pouring your gel

28

Pour running gel first. Contains Tris buffer at pH 8.0. The percent of acrylamide may be adjusted for better resolution of proteins (5-15%)Pour ethanol or distilled water on top of gel for even

polymerization.Leave enough room to pour stacking gel, about one forth

of total gel.

Asheesh Pandey

Pouring gel continues

29

After the running gel has polymerized, the gel is washed with distilled water to remove any debris on the gel to give a good interface between stacking and running gels. The stacking gel is pour. It contains Tris buffer at pH 6.8. 5% acrylamide for maximum porosityDeposits proteins on stacking and running gel interface

which concentrates the proteins and provides better resolution.

Insert comb to form wells at an angle to prevent air bubbles.

Asheesh Pandey

Loading gel

30

Remove comb and wash wells out with running buffer.

Best to use loading tips (Hamilton syringes also work) to load samples.

Start at the bottom of well and work your way up the well.

Glycerol in the loading buffer will keep sample in well.

Optional- Running buffer left in wells or wells empty.

Asheesh Pandey

Running Gel

31

After loading samples, added running buffer to upper reservoir and lower reservoir. Hint. Add upper reservoir first to detect leaks.

Running buffer provides the ions to conduction the current through the gel.

SDS makes proteins negatively charged that attaches the proteins to the anode.

Therefore in electrophoresis, the current must run from cathode (negatively charged, black) to the anode (positive charged, red).

Asheesh Pandey

Considerations with PAGE

32

Preparing and Pouring GelsDetermine pore size

Adjust total percentage of acrylamideVary amount of crosslinker

Remove oxygen from mixtureInitiate polymerization

Chemical methodPhotochemical method

Asheesh Pandey

Considerations with PAGE

33

Analysis of GelStaining or autoradiography followed by

densitometry Blotting to a membrane, either by capillarity

or by electrophoresis, for nucleic acid hybridization, autoradiography or immunodetection

Asheesh Pandey

SDS Gel Electrophoresis

34

In SDS separations, migration is determined not by intrinsic electrical charge of polypeptides but by molecular weight

Sodium dodecylsulfate (SDS) is an anionic detergent which denatures secondary and non–disulfide–linked tertiary structures by wrapping around the polypeptide backbone. In so doing, SDS confers a net negative charge to the polypeptide in proportion to its length

When treated with SDS and a reducing agent, the polypeptides become rods of negative charges with equal “charge densities" or charge per unit length.

Asheesh Pandey

SDS Gel Electrophoresis

35 Asheesh Pandey

Continuous and Discontinuous Buffer Systems

36

A continuous system has only a single separating gel and uses the same buffer in the tanks and the gel

In a discontinuous system a nonrestrictive large pore gel, called a stacking gel, is layered on top of a separating gel

The resolution obtainable in a discontinuous system is much greater than that obtainable in a continuous one. However, the continuous system is a little easier to set up

Asheesh Pandey

Continuous and Discontinuous Buffer Systems

37 Asheesh Pandey

Commonly used protein stains

38

Stain Detection limit

Ponceau S 1-2 g

Amido Black 1-2 g

Coomassie Blue 1.5 g

India Ink 100 ng

Silver stain 10 ng

Colloidal gold 3 ng

Asheesh Pandey

Chromogenic stains

39 Asheesh Pandey

Fluorescent stains

40 Asheesh Pandey

Determining Molecular Weights of Proteins by SDS-PAGE

Run a gel with standard proteins of known molecular weights along with the polypeptide to be characterized A linear relationship exists between the

log10 of the molecular weight of a polypeptide and its Rf

Rf = ratio of the distance migrated by the molecule to that migrated by a marker dye-front The Rf of the polypeptide to be

characterized is determined in the same way, and the log10 of its molecular weight is read directly from the standard curve

41Asheesh Pandey

Blotting

42

Blotting is used to transfer proteins or nucleic acids from a slab gel to a membrane such as nitrocellulose, nylon, DEAE, or CM paper

The transfer of the sample can be done by capillary or Southern blotting for nucleic acids (Southern, 1975) or by electrophoresis for proteins or nucleic acids

Asheesh Pandey

Isoelectric PointThere is a pH at which there is no net

charge on a protein; this is the isoelectric point (pI).

Above its isoelectric point, a protein has a net negative charge and migrates toward the anode in an electrical field.

Below its isoelectric point, the protein is positive and migrates toward the cathode.

43Asheesh Pandey

Isoelectric Focusing

44

Isoelectric focusing is a method in which proteins are separated in a pH gradient according to their isoelectric points

Focusing occurs in two stages; first, the pH gradient is formed

In the second stage, the proteins begin their migrations toward the anode if their net charge is negative, or toward the cathode if their net charge is positive

When a protein reaches its isoelectric point (pI) in the pH gradient, it carries a net charge of zero and will stop migrating

Difference 2-D?

Asheesh Pandey

Two-Dimensional Gel Electrophoresis

45

Two-dimensional gel electrophoresis is widely used to separate complex mixtures of proteins into many more components than is possible in conventional one-dimensional electrophoresis

Each dimension separates proteins according to different properties

Asheesh Pandey

Two-dimensional gel electrophoresis(2-D electrophoresis )

In the first dimension, proteins are resolved in according to their isoelectric points (pIs) using immobilized pH gradient electrophoresis (IPGE), isoelectric focusing (IEF), or non-equilibrium pH gradient electrophoresis. In the second dimension, proteins are separated according to their approximate molecular weight using sodium dodecyl sulfate poly-acrylamide-electrophoresis (SDS-PAGE).

46 Asheesh Pandey

O’Farrell 2D Gel System

47

The first dimension tube gel is electrofocused

The second dimension is an SDS slab gel

The analysis of 2-D gels is more complex than that of one-dimensional gels because the components that show up as spots rather than as bands must be assigned x, y coordinates

Asheesh Pandey

• DIGE can be done in one-or two-dimensions. Same principle.

• Requires fluorescent protein stains (up to three of these), a gel box, and a gel scanner.

• Dyes include Cy2, Cy3 and Cy5 (Amersham system).

• These have similar sizes and charges, which means that individual proteins move to the same places on 2-D gels no matter what dye they are labeled with.

• Detection down to 125 pg of a single protein.

DIfference Gel ElectrophoresisDIfference Gel Electrophoresis

DIGEDIGE

48 Asheesh Pandey

49 Asheesh Pandey

• After running the gels, three scans are done to extract the Cy2, Cy3, and Cy5 fluorescence values.

• Assuming the Cy2 is the internal control, this is used to identify and positionally match all spots on the different gels.

• The intensities are then compared for the Cy3 and Cy5 values of the different spots, and statistics done to see which ones have significantly changed in intensity as a consequence of the experimental treatment.

50 Asheesh Pandey

• Labeling slightly shifts the masses of the proteins, so to cut them out for further analysis, you first stain the gel with a total protein stain.

• SYPRO Ruby is used for this purpose (Molecular Probes).

• When designing 2-D DIGE experiments, the following recommendations should be considered:

1. Inclusion of an internal standard sample on each gel. These can comprise a mixture of known proteins of different sizes, or simply a mixture of unknown proteins (one of your samples).

2. Use of biological replicates.3. Randomization of samples to produce unbiased

results.51 Asheesh Pandey

BLOTTING

1. SOUTHERN BLOT

2. NORTHERN BLOT

3. WESTERN BLOT

Blotting: History

53

Southern Blotting is named after its inventor, the British biologist Edwin Southern (1975)

Other blotting methods (i.e. western blot,

WB, northern blot, NB) that employ similar principles, but using protein or RNA, have later been named in reference to Edwin Southern's name.

Asheesh Pandey

SOUTHERN BLOTTING ?

54

Experimental procedure DNA is extracted from cells, leukocytes.

DNA is cleaved into many fragments by �restriction enzyme (BamH1, EcoR1 etc)

Asheesh Pandey

VNTR

STR

Microsatelite

Macrosatelite

RFLP

AFLP

55 Asheesh Pandey

56

The resulting fragments are separated on the basis of size by electrophoresis.

The large fragments move more slowly than the

smaller fragments.

The lengths of the fragments are compared with band of relative standard fragments of known size.

Asheesh Pandey

57

The DNA fragments are denatured and transferred to nitrocellulose membrane (NYTRAN) for analysis.

DNA represents the individual's entire genome, the enzymic digest contains a million or more fragments.

The gene of interest is on only one of these pieces of DNA.

Asheesh Pandey

58

DNA segments were visualized by a nonspecific technique, they would appear as an unresolved blur of overlapping bands.

To avoid this, the last step in Southern blotting uses a

probe to identify the DNA fragments of interest.

Asheesh Pandey

59

Southern blot analysis depend on the specific restriction endonuclease

The probe used to visualize the restriction fragments.

Asheesh Pandey

•Labeled material to detect a target.

•For DNA: 20-30 nucleotides, complementary to a region in the gene

•Methods of labeling:

•Non-radioactive e.g. Biotin•Radioactive e.g. 32P

•Sensitive•Relatively cheap•HazardousYou should follow the radioactive waste disposal regulations.

•Sensitive•Relatively expensive

Target DNA

ProbeBiotin Avidin

*

Target DNA

Probe*

Probes

60 Asheesh Pandey

The binding between ss labeled probe to a complementary nucleotide sequence on the target DNA.Degree of hybridization depends on method of probe labeling (radioacitve or non-radioactive system e.g. biotin-avidin.

Hybridization

61 Asheesh Pandey

62

Detection of mutations The presence of a mutation affecting a restriction site

causes the pattern of bands to differ from those seen with a normal gene.

A change in one nucleotide may alter the nucleotide sequence so that the restriction endonuclease fails to recognize and cleave at that site (for example, in Figure, person 2 lacks a restriction site present in person 1).

Asheesh Pandey

StepsDigestion of genomic DNA (w/ ≥ one RE) DNA fragments

Size-separation of the fragments (standard agarose gel electrophoresis)

In situ denaturation of the DNA fragments (by incubation @ ↑temp)

Transfer of denatured DNA fragments into a solid support (nylon or nitrocellulose).

Hybridization of the immobilized DNA to a labeled probe (DNA, RNA)

Detection of the bands complementary to the probe (e.g. by autoradiography)

Estimation of the size & number of the bands generated after digestion of the genomic DNA w/ different RE placing the target DNA within a context of restriction sites)

63 Asheesh Pandey

METHODS OF TRANSFER

Downward Capillary Transfer

Upward Capillary Transfer

Simultaneous Transfer to Two Membranes

Electrophoretic Transfer

Vacuum Transfer

64 Asheesh Pandey

Example of TransferUpward Capillary Transfer

WeightGlass Plate

Whatman 3MM paper

Gel

Paper towels

Membrane (nylon or nitrocellulose)

Whatman 3MM paper

Transfer buffer65 Asheesh Pandey

Buffer drawn from a reservoir passes through the gel into a stack of paper towels

DNA eluted from the gel by the moving stream of buffer is deposited onto a membrane

weight tight connection

66 Asheesh Pandey

Northern BlottingNorthern Hybridization

A northern blot is a method routinely used in molecular biology for detection of a specific RNA sequence in RNA samples.

The method was first described in the seventies (Alwine et al. 1977, 1979)

It is still being improved (Kroczek 1993), with the basic steps remaining the same

67 Asheesh Pandey

Basis Steps of NB1. Isolation of intact mRNA2. Separation of RNA according to size (through a

denaturing agarose gel e.g. with Glyoxal/formamide)Transfer of the RNA to a solid supportFixation of the RNA to the solid matrixHybridization of the immobilized RNA to probes complementary to the sequences of interestRemoval of probe molecules that are nonspecifically bound to the solid matrixDetection, capture, & analysis of an image of the specifically bound probe molecules.

68 Asheesh Pandey

ApplicationsStudy of gene expression in eukaryotic cells:

To measure the amount & size of RNAs transcribed from eukaryotic genes

To estimate the abundance of RNAs Therefore, it is crucially important to equalize

the amounts of RNA loaded into lanes of gels

69 Asheesh Pandey

Examples of methods to equalize the amounts of RNA loaded into lanes of gels

OD260

Use of housekeeping gene (endogenous constitutively-expressed gene): Normalizing samples according to their content of mRNAs of this housekeeping gene

70 Asheesh Pandey

Western Blotting“Immunoblotting”= electrophoretic transfer of proteins from gels to membranes

71 Asheesh Pandey

WB: Definition

Blotting is the transfer of separated proteins from the gel matrix into a membrane, e.g., nitrocellulose membrane, using electro- or vacuum-based transfer techniques.

Towbin H, et al (1979). "Electrophoretic transfer of proteins Towbin H, et al (1979). "Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and from polyacrylamide gels to nitrocellulose sheets: procedure and some applications.". some applications.". Proc Natl Acad Sci U S A.Proc Natl Acad Sci U S A. 76 (9): 4350–4354 76 (9): 4350–4354

72 Asheesh Pandey

Applications & AdvantagesApplications:

To determine the molecular weight of a protein (identification)

To measure relative amounts (quantitation) of the protein present in complex mixtures of proteins that are not radiolabeled (unlike immunoprecipitation)Advantages:

WB is highly sensitive techniqueAs little as 1-5 ng of an average-sized protein can be detected by WB

73 Asheesh Pandey

74

Western blotting

The main steps of blotting technique in a chronological order will be as follows:

BlockingProbing with the specific antibody(ies)WashDetection WashingX-ray (Gel Documentation System)

Asheesh Pandey

Electrophoretic Transfer: An Overview

Important Issue:Important Issue:Where to put the gel and the membrane relative to the Where to put the gel and the membrane relative to the

electroblotting transfer electrodes?electroblotting transfer electrodes?75 Asheesh Pandey

Direction of Transfer

Perpendicularly from the direction of travel of proteins through the separating gel

Gel

Membrane

Probe with specific Ab

76 Asheesh Pandey

Factors Affecting Transfer Efficiency1. The Composition of the gel2. Whether there is complete contact of the

gel with the membrane3. The position of the electrodes4. The transfer time5. The size & composition of proteins6. The field strength7. The presence of detergents

77 Asheesh Pandey

WB Procedure; Briefly…12

3 478 Asheesh Pandey

Direct Detection Method

79 Asheesh Pandey

Indirect Detection Method

80 Asheesh Pandey

WB: examples of used substrates

81 Asheesh Pandey

Chemiluminescent substrates

82 Asheesh Pandey

Enhanced ChemiFluoresenct (ECF) WB Detection

83 Asheesh Pandey

Western Blotting Procedure; Illustrated

85

Blocking of Blot

Several measures should be followed to decrease the nonspecific reactions to a minimum, i.e., increasing the signal to noise ratio.

Blocking step is the incubation of the membrane with solution containing BSA or fat-free milk or casein for a sufficient time with shaking.

Asheesh Pandey

Steps of WB

86

For Direct Transfer, choices are:

Asheesh Pandey

87

Primary Antibody labeling

The immobilized proteins on the surface of the membrane can be detected using a specific, labeled antibody.

Labeling of the antibody can be performed using a radioactive or non- radioactive method.

Asheesh Pandey

88

Primary Antibody probing

The blot is first incubated with a primary antibody followed by the addition of a labeled secondary antibody that has species specificity for the primary one.

For example, probing of the membrane using mouse primary antibody and anti- mouse secondary antibody.

Asheesh Pandey

89

Detection and interpretation

A prestained MW standard is included in a separate lane during electrophoresis to allow the identification of the MW of the target protein.

Similar to the analysis of electrophoresis results on a gel, the data on the membrane can be quantitatively analyzed using gel documentation system.

Asheesh Pandey

90

Detection and interpretation (continue)

Quantification of a specific protein band can be achieved by densitometry and integrating the areas under the peaks.

Several gel documentation systems are commercially available that can be useful for analysis of results from the gel or membranes.

Asheesh Pandey

Comparison between WB & SB.

91

Similarities:Electrophoretically separated components (proteins in

WB & DNA in SB), are transferred from a gel to a solid support and probed with reagents that are specific for particular sequences of AA (WB) or nucleotides (SB).

Asheesh Pandey

References

92

Lippincott, Illustrated review of Biochemistry, 4th edition

Molecular Cloning: A Laboratory Manual, J Sambrook, EF Fritsch, T Maniatis

Catalogues of some commercial companies

Asheesh Pandey