SDS-PAGE and Western Analysis

SDS-PAGEpurposes

To separate protein molecules on the basis of molecular weight and

To determine the molecular weights of unknowns by comparison to standards

SDS-PAGE preparation of cell extract Lyse cells in RIPA buffer containing inhibitors of

both proteases and phosphatases. Centrifuge lysate to remove membranous cellular

debris. Determine protein concentration of the lysate in

g/l. Example: Bradford method - Binding of Coomassie

Blue dye by proteins in solution and comparison to standards of known concentration

preparation of cell extract components of the lysis buffer

50 mM Tris-HCl, pH 8.0 – pH friendly to most proteins

150 mM NaCl, isotonic saline Detergents – disrupt lipid bilayers; aid in

solubilizing hydrophobic proteins NP-40 (non-ionic, good solubilization, weakly

denaturing) Deoxycholate (a bile acid, ionic, moderately

denaturing) SDS (synthetic, ionic, excellent solubilization,

strongly denaturing)

~NP-40

Components of the lysis buffer (cont’d)

Dithioerythritol – a reducing agent Prevents inappropriate oxidation of reduced

cysteines to disulfide bonds. Prevents covalent aggregation and precipitation of

proteins that are not covalently linked in vivo. Especially important for native gel electrophoresis.

(See Slide #12. DTT in the lysis buffer is insufficiently concentrated to disrupt the disulfide bonds which form the natural structure of some proteins, but is sufficiently concentrated to prevent inappropriate disulfide bonds from forming.

Protease inhibitors Prevent protein degradation and thereby

allow more accurate determination of molecular weight Examples

PMSF – inhibits serine proteases• phenylmethanesulfonylfluoride

Leupeptins• tripeptides produced by various species of Actinomycetes

• L-leucyl-L-leucyl-Dl-argininal

– modified at NH –terminus by acetyl or propionyl Aprotinin

• Found in pancreas and lung, among other tissues

• Natural inhibitor of various extra- and intracellular proteases

Components of the lysis buffer (cont’d)

PMSF

Aprotinin

Phosphatase inhibitors Inhibitors prevent enzymatic removal of phosphates

from phosphorylated proteins during extract preparation

Phosphorylated and dephosphorylated proteins migrate differently during SDS-PAGE

Useful information can be gained by knowing whether or not a protein is phosphorylated in vivo in given cells under specific conditions

Examples of general phosphatase inhibitors NaF Na3VO4

Components of the lysis buffer (cont’d)

SDS-PAGE preparation of sample for loading

Major components of the sample loading “buffer” SDS DTT Tracking dye Glycerol

SDS-PAGE preparation of sample for loading

Major purposes of boiling in loading buffer are to denature and coat proteins with SDS

proteins bind SDS with similar ratios of detergent to protein mass (-) charge on dodecyl sulfate ions ~ = charge/mass ratio for all

proteins, so separation is on the basis of size

reduce disulfide bonds using DTT (or -mercaptoethanol) Causes disulfide bonded peptides to become independent (see

next slide. Good for determining size of disulfide-bonded subunits

HeatExcess DTT

2

+

2H

H

H

Reduction by monovalent mercaptans

Reduction by divalent mercaptans

-mercaptoethanol

DTTdithioerythritol

dithioerythritol(reduced)

dithioerythritol(oxidized)

Dye is included to monitor migration during PAGE Bromphenol blue

Glycerol is included to make sample denser than running buffer minimizes diffusion during loading

preparation of sample for loading

SDS-PAGE gel system (Note features in red!)

Discontinuous Two gel layers with different polyacrylamide

concentrations A different buffer for each of the two parts of the gel Yet a third buffer as the running buffer

Stacking (concentrating) gel 4% acrylamide (36.5:1, acryl/bis) 125 mM Tris-H+Cl-, pH 6.8, 0.1% SDS

Resolving (separating) gel 10% acrylamide (36.5:1, acryl/bis) 425 mM Tris-H+Cl-, pH 8.8, 0.1% SDS

Running buffer 25 mM Tris-H+Cl-, pH 8.3, 192 mM glycine, 1% SDS

Why use a discontinuous gel and buffer system for SDS-PAGE?

Purpose of the stacking gel: to concentrate all the proteins in the sample into a thin band at the top of the resolving gel Makes it possible to use a dilute sample

Purpose of the resolving gel: to separate the proteins on the basis of size.

The next set of slides will address how the stack works. Following that will be a set of slides on the resolving

system.

stacking gel

Concentrates proteins because it Has large pores (4%), so proteins of all sizes

move easily through the pores of the stacking gel until they meet a frictional barrier at the top of the resolving gel, with its smaller pores (10%).

But that’s not the only way the proteins are concentrated!

Clever design of a discontinuous buffer system increases the concentrating effect of the stacking gel on the proteins in the sample!

stacking gel Concentrates proteins also because it

uses the stacking and running buffers to form a voltage gradient protein mobility Stacking gel buffer is of

• Low salt concentration• Cl- = leading ion because it is small, negatively charged,

and moves quickly through gel Running buffer ion is primarily

• glycine = trailing “ion”, which at pH 6.8 is nearly neutral A region of low ionic strength quickly develops between Cl-

and glycine, generating a voltage gradient. Large, negatively charged proteins are left to constitute

most of the molecular current, and move quickly to the bottom of the stacking gel.

pH 6.8, neutral

Notice the progression of Cl-, negatively charged proteins of different sizes (P-), and mostly neutral glycine (G) in the next three slides.

Cl-Cl-

Cl-

Cl-

Cl-

Cl-

Cl-Cl-

Cl- Cl-

Cl-Cl-

Cl-Cl-

Cl-Cl-Cl-

Cl-

Cl- Cl-Cl-

Cl-Cl-

Cl-

Cl-Cl-Cl-

Cl- Cl-

Cl-

Cl-

Cl-Cl- Cl-

Cl- Cl-Cl-

Cl-

Cl-

Cl-

Cl- Cl-Cl-

Cl-

Cl- Cl-

Cl-

Cl-

Cl-Cl- Cl-Cl- Cl-

Cl-

Cl- Cl-

P-P-P-

P--

P-

P--

G G G G G

Cl- Cl- Cl- Cl- Cl-

Cl-

Cl-Cl-

Cl- Cl-

Cl-Cl-

Cl-Cl-

Cl-Cl-Cl-

Cl-

Cl- Cl-Cl-

Cl-Cl-

Cl-

Cl-Cl-Cl-

Cl- Cl-

Cl-

Cl-

Cl-Cl- Cl-

Cl- Cl-Cl-

Cl-

Cl-

Cl-

Cl- Cl-Cl-

Cl-

Cl- Cl-

Cl-

Cl-

Cl-Cl- Cl-Cl- Cl-

Cl-

Cl- Cl-

P-P-P-

P--

P-

P--

G G G G G

Cl- Cl- Cl- Cl- Cl-

Cl-

Cl-Cl-

Cl- Cl-

Cl-Cl-

Cl-Cl-

Cl-Cl-Cl-

Cl-

Cl- Cl-Cl-

Cl-Cl-

Cl-

Cl-Cl-Cl-

Cl- Cl-

Cl-

Cl-

Cl-Cl- Cl-

Cl- Cl-Cl-

Cl-

Cl-

Cl-

Cl- Cl-Cl-

Cl-

Cl- Cl-

Cl-

Cl-

Cl-Cl- Cl-Cl- Cl-

Cl-

Cl- Cl-

P- P-P- P--P- P--

G G GG

G

Cl- Cl- Cl- Cl- Cl-

Cl-

Cl-Cl-

Cl- Cl-

Cl-Cl-

Cl-Cl-

Cl-Cl-Cl-

Cl-

Cl- Cl-Cl-

Cl-Cl-

Cl-

Cl-Cl-Cl-

Cl- Cl-

Cl-

Cl-

Cl-Cl- Cl-

Cl- Cl-Cl-

Cl-

Cl-

Cl-

Cl- Cl-Cl-

Cl-

Cl- Cl-

Cl-

Cl-

Cl-Cl- Cl-Cl- Cl-

Cl-

Cl- Cl-

P- P-P- P--P- P--

GG

G

G

G

G G

On to the resolving gel . . .

The resolving gel separates proteins as a function of percentage acrylamide ratio of acrylamide to bis extent of difference in size between the proteins

being resolved

The resolving gel

Has a higher [ion] (0.425 M Tris-H+Cl-) than the stacking gel (0.125 M Tris-H+Cl-) so Proteins contribute less to the total ionic current than

they did in the stacking gel, and as a result the mobilities of proteins and differences in mobilities

among proteins of different sizes become more apparent.

Resolving gel

In addition, the resolving gel has a higher pH (8.8) than the stacking gel (6.8) so glycine takes on a more negative charge, thereby the

increasing the total ion concentration and because it is small, moves ahead of the proteins.

So proteins move slowly through the gel and are resolved by friction on the basis of size.

pH 6.8, neutral pH 8.8, negatively charged

H2

Cl- Cl- Cl- Cl- Cl-

Cl-

Cl-Cl-

Cl- Cl-

Cl-Cl-

Cl-Cl-

Cl-Cl-Cl-

Cl-

Cl- Cl-Cl-

Cl-Cl-

Cl-

Cl-Cl-Cl-

Cl- Cl-

Cl-

Cl-

Cl-Cl- Cl-

Cl- Cl-Cl-

Cl-

Cl-

Cl-

Cl- Cl-Cl-

Cl-

Cl- Cl-

Cl-

Cl-

Cl-Cl- Cl-Cl- Cl-

Cl-

Cl- Cl-

P- P-P-P--

P-P--

G-

G

G

G

G

G G

G

G-

G-

G-

Cl- Cl- Cl- Cl- Cl-

Cl-

Cl-Cl-

Cl- Cl-

Cl-Cl-

Cl-Cl-

Cl-Cl-Cl-

Cl-

Cl- Cl-Cl-

Cl-Cl-

Cl-

Cl-Cl-Cl-

Cl- Cl-

Cl-

Cl-

Cl-Cl- Cl-

Cl- Cl-Cl-

Cl-

Cl-

Cl-

Cl- Cl-Cl-

Cl-

Cl- Cl-

Cl-

Cl-

Cl-Cl- Cl-Cl- Cl-

Cl-

Cl- Cl-

P- P-P-

P--

P-

P--

G-

G

G

G

G

G G

G

G- G-

G-G-

G-

G G G

Immunoblotting Electrophoretic transfer is used to move proteins

out of gel onto membrane in equivalent relative positions for subsequent antibody probing Nitrocellulose membrane is most commonly used

Nature of bonding between proteins and nitrocellulose is not known.

Other membrane types can be used PVDF, nylon, charged nylon Each has advantages and disadvantages

Electrophoretic transfer Fast Usually complete

Immunoblotting

Equilibration of gel in transfer buffer (25 mM Tris, 192 mM glycine, 20% v/v methanol) Removes excess electrophoresis buffer salts from gel.

The higher the ionic strength during electrophoretic transfer, the more heat is generated.

Excess heat is a safety hazard.

Immunoblotting Equilibration of gel in transfer buffer (25 mM Tris, 192

mM glycine, 20% methanol) Removes detergents from gel.

Detergents interfere with binding of protein to membrane. 0.01 - 0.02% SDS in transfer buffer can improve transfer

out of gel. Equilibrates the gel with methanol.

Methanol is required for binding of proteins to nitrocellulose.

• Methanol shrinks the gel.– Gel must be allowed to complete shrinking before blot

sandwich is assembled.– Shrinkage may interfere with transfer of larger proteins.

After transfer Check for success of transfer

See if the colored protein standards are on the membrane, and/or

Stain the proteins on the blot with stain that does not interfere with immunological probing. Example: Ponceau S

• Can also be done after immunoprobing

Immunoprobing Choice of blocking agents Choice of 1o Antibody Choice of 2o Antibody Choice of antibody concentrations Visualization system

agent

Qualities of Several Blocking Agents

Tween

Factors to Consider when Choosing the Type of 1o Antibody Preparation to Use in a Western

Immunoprobing Choice of 2o Antibody

2o Ab must recognize the common portion of the 1o Ab. 1o Ab is raised in a given animal

e.g., mouse i.e., mouse was immunized by injection with the antigen

• e.g., denatured p53

2o Ab must be raised in a different animal e.g., goat or rabbit i.e., animal immunized by injection with Ig that is of the

• same class (IgG) as 1o Ab , but of different irrelevant specificities

Immunoprobing Choice of antibody concentrations

Use as little 1o and 2o Ab as possible to generate detectable signal. Minimization of [Ab] cleaner results lower cost

Too much of either 1o or 2o Ab background non-specific binding

Various 1o and 2o Ab concentrations must be tried to optimize signal minimize noise

Western Immunoprobing Enzymatic detection systems

Alkaline phosphatase Horseradish peroxidase

Direct vs. indirect detection Note, the meanings of direct vs. indirect in

immunoprobing on a blot is NOT the same as for an ELISA.

Immunoprobing Enzymatic detection systems

Horseradish peroxidase catalyzes the rx . . Luminol + H2O2 light + 3-aminophthalate + N2 + H2O

Western Immunoprobing Direct detection - 1o Ab is enzyme-conjugated

Fewer background bands and background than indirect detection, but

Less sensitive and Requires that 1o Ab of every different specificity be purified

and conjugated to enzyme Indirect detection - 2o Ab is enzyme-conjugated

More sensitive than direct Less expensive than direct - one preparation of labeled 2o Ab

can recognize all 1o Abs from the same species. HRP conjugated rabbit anti-mouse IgG can be used in a

Western for any protein recognized by a 1o Ab raised in mouse.

Comparing your Western positive band to size standards

Make a standard curve on semi-log paper,plotting the distance traveled against the known sizes of the standards.

Measure the distance traveled by the protein giving you a positive band on your Western.

Determine the size of your protein from the standard plot.

Myosin, 202 kd

-galactosidase, 133 kd

BSA, 71 kd

Carbonic anhydrase, 41.8 kd

Soybean trypsin inhibitor, 30.6 kd

Lysozyme, 17.8 kd

Aprotinin, 6.9 kd

MWs adjusted to account for masses of covalently bound dyes

(red)

= distance traveled

7% resolving gel

10% resolving gel

Predict the distance traveled by p53 in each of the two gels for which the standards are plotted at right.