Protein electrophoresis:

Introduction to SDS-PAGE

Aim:

-Separation of proteins in an electric field by electrophoresis.

Purposes:

-Estimation of molecular masses

-Relative abundances of major proteins in a sample

SDS-PAGE = Sodium Dodecyl Sulfate PolyAcrylamide Gel

Electrophoresis.

Uses a polyacrylamide gel as a support medium and SDS to

denature proteins.

Also called the Laemmli method.

Laemmli UK, Nature 1970, 227 (5259):680-5. Cleavage of structural proteins during the assembly of the

head of bacteriophage T4..

Laemmli UK, Nature 1970, 227

(5259):680-5. Cleavage of structural

proteins during the assembly of the

head of bacteriophage T4..

http://www.bio-rad.com/LifeScience/pdf/Bulletin_1156.pdf

SDS is an anionic detergent.

A polypeptide chain binds SDS in molar proportion to its mass.

SDS denatures most complex structures of proteins. It is also

attracted toward the positively-charged anode.

About 1 SDS binds per 2 AA.

Polyacrylamide gels restrain larger molecules from migrating as fast

as smaller ones.

The separation of proteins depends almost entirely on the

differences between the molecular masses of polypeptides.

In a uniform gel, the migration distance of a protein (Rf) is

proportional to the -log of its mass.

Proteins of known masses are run along with the unknowns. This

allows to plot “Rf vs. mass”, and to estimate the masses of unknown

proteins.

Stacking gel

Separating (or

Resolving) gel

-

+

Lighter protein

Heavier protein

Polyacrylamide gels for SDS-PAGE

Important parameters:

-relative percentages of acrylamide and crosslinker (e.g. bis)

-gel dimensions

“Manual” preparation requires casting two different layers of

acrylamide between glass plates.

Lower layer: separating gel.

Upper layer: stacking gel with sample wells. Stacks proteins into

micrometer thin layers.

SDS Gels

-A 7% bis in acrylamide gel separates proteins with masses between

45 and 200 kDa (no resolution below 45 kDa).

-A denser gel, e.g. 14%, resolves smaller polypeptides. It would not

separate proteins above 60 kDa.

-To analyze the entire profile of a sample containing heavy and light

polypeptides: run two gels, or a density gradient gel.

ww2.chemistry.gatech.edu

Running Gel:

Buffer stock solution: 0.4% SDS, 0.5 M Tris-HCl @ pH 6.8.

Buffer, linker and acrylamide stock solutions are mixed to obtain from 7

to 15% of bisacrylamide.

Acrylamide polymerizes in the absence of O2: the mixture is placed

under vacuum for 5 min.

Then polymerization occurs quickly. The solution is poured into a

cassette.

The gel mix is poured, then overlaid with water-saturated butanol.

This will produce a smooth, leveled surface to keep bands straight

and uniform.

Stacking gel

•Stackers have 3-4.5% acrylamide (low density) to enable very large

proteins to run.

•At polymerization, the mix is poured into the cassettes on top of the

separating gel.

•A comb is inserted and adjusted evenly.

http://www.manufacturingchemist.com/news/article_page/title/46647

proteabio.com

Protein + sample denaturation

Protein denaturation prior to SDS-PAGE

Sample is mixed with 2% SDS, 20% glycerol, 20 mM Tris-Cl, pH 8, 2

mM EDTA, dithiothreitol (DTT), and bromophenol blue as a tracking

dye.

Roles of denaturing components

EDTA chelates divalent cations, then reduces the activity of enzymes

that require Ca2+ and Mg2+ ions.

Glycerol makes the sample denser than the buffer: sample will

remain at the bottom of a well rather than float out.

Dye: tracks the progress of electrophoresis.

SDS: adds negative charge to the amino acids. Proteins are

“straightened”, thus functionless.

DTT breaks covalent disulfide bonds.

Cystine contains a sulfhydryl (-SH) group that spontaneously forms

a disulfide bond (-S-S-) with another sulfhydryl group under normal

intracellular conditions.

DTT is a strong reducing agent for disulfide bonds.

Amounts to loadA typical mini-gel well holds 10 µL of a 2 mg/mL solution of

denatured protein (0.02 mg or 20 µg).

SDS-PAGE yields reproducible results, but provides low

accuracy for MW determination.

Running gels

The anode (+) must be connected to the bottom chamber and the

cathode (-) to the top chamber.

Gels are run at a voltage (typically 150 V) that will bring the

tracking dye to the bottom as quickly as possible without

overheating the gels.

Disassembly and staining

When the dye front is near the

bottom of the gel, the run is

stopped.

Plates are separated and the

gel is dropped into a staining

dish.

Staining usually requires

incubation overnight, with

agitation.

Staining protein gels

Commonly used: 0.1% Coomassie Blue dye in 50% methanol, 10%

glacial acetic acid.

Acidified methanol precipitates the proteins.

The dye inflitrates the entire gel, however only sticks permanently to

proteins.

Excess dye is washed out by 'destaining' with acetic acid/methanol,

also with agitation.

Gels can be dried down, cut or photographed for later analysis and

documentation.

http://upload.wikimedia.org/wikipedia/commons/5/55/Coomassie_Brilliant_Blue_G-250.svg

Steps to analyze the gel

Calibrate the gel using standards of known molecular mass

Estimate molecular mass for each band of interest

Note differences in intensity of staining that reflect abundance of

individual proteins

Note unusual patterns that might indicate incomplete denaturation,

degradation, etc.

Estimate apparent molecular mass for unknowns

The relationship between mass and Rf is logarithmic. Data are

interpolated from the standard curve.

High molecular mass:

-myosin (205,000) - from rabbit muscle

-beta-galactosidase (116,000) - from Escherichia coli

-phosphorylase B (97,400) - from rabbit muscle

-bovine serum albumin (66,000)

-egg albumin (45,000)

-carbonic anhydrase (29,000) - from bovine erythrocytes

Low molecular mass

-bovine serum albumin 66,000

-egg albumin 45,000

-glyceraldehyde-3-phosphate dehydrogenase (36,000) - from

rabbit muscle

-carbonic anhydrase (29,000)

-trypsinogen (24,000) - from bovine pancreas

-soybean trypsin inhibitor (20,100)

-alpha-lactalbumin (14,200) - from bovine milk

205k

116k

97.4k

66k

45k

36k

29k

24k

20.1k

Measuring relative mobility of protein bands

Relative mobility is the distance migrated by a band divided by the

distance migrated by the dye front.

It is used to compare the migration of a protein from gel to gel,

regardless of the physical length of the gel or duration of

electrophoresis.

How gel concentration affects relative mobility

Acrylamide concentration determines the cut-off at the low end.

All polypeptides below a minimum mass run with the tracking dye.

A gel with low density resolves larger polypeptides while cutting off

lighter ones.

A gel of higher density will reveal smaller polypeptides, while

compressing and possibly distorting the larger ones.

3 = phosphorylase B (92,000)

Summary

SDS-PAGE: advantages

Relatively inexpensive

Low voltage used: easy to handle

Visual result

Makes comparison of samples easy

Possible to cut protein band from gel for further use

Disadvantages:

Mass determination not accurate

Results subject to experience of experimentalist

Stain may be difficult to remove for further use

Gel storage in liquid

Quantitation not accurate

1. a) The following standard proteins (table) serve as MW markers during SDS-PAGE separations. Draw a hypothetical gel showing the migration pattern of these proteins given an optimal gel composition for complete separation.

b) What would the separation patterns look like if a lower than optimal % acrylamide was used, or a higher percentage?

c) Discuss the effects of post-translational modifications (phosphorylation, glycosylation) on the mass measurements of proteins by SDS-PAGE.

2.

a) In SDS-PAGE, some gels are prepared with a gradient. Which parameter is setup as a

gradient and how is this done?

b) Explain the effect of a gel gradient on the separation of low and high MW proteins.