1. SDS-PAGE

2. SDS-PAGE purposes
To separate protein molecules on the basis of molecular weight
May then be electroblotted for immunoanalysis
To determine the molecular weights of unknowns by comparison to standards

3. HRP
+ ECL substrate  light
HRP conjugated 2o antibody
1o antibody
Proteins blotted to membrane from SDS-PAGE gel
What’s wrong?
Membrane

4. 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 in g/l of the lysate.
Example: Bradford method - Binding of Coomassie Blue dye by proteins in solution and comparison to standards of known concentration

5. 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)

6. ~NP-40

7. Components of the lysis buffer (cont’d)
Dithiothreitol – a reducing agent
Prevents inappropriate oxidation of reduced cysteines to disulfide bonds, and thereby
Prevents covalent aggregation and precipitation of proteins that are not covalently linked in vivo.
DTT is 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.)

8. Components of the lysis buffer (cont’d)
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-D-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

9. PMSF

10. Aprotinin

11. Components of the lysis buffer (cont’d)
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

12. SDS-PAGE preparation of sample for loading
Major components of the sample loading “buffer”
SDS
DTT
Tracking dye
Glycerol

13. SDS-PAGE preparation of sample for loading
Major purposes of boiling in loading buffer are to
denature and coat proteins with SDS
All 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

14. 2 2 H H
Heat
Excess DTT  + H

15. Reduction by monovalent mercaptans
-mercaptoethanol
Reduction by divalent mercaptans
dithiothreitol
(reduced)
dithiothreitol
(oxidized)
DTT
dithiothreitol

16. preparation of sample for loading
Dye is included to monitor migration during PAGE
Bromphenol blue
Glycerol is included to make sample denser than running buffer
minimizes diffusion during loading

17. 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
And 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 base; 192 mM glycine, pH 8.3; 1% SDS

18. 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.

19. 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!

20. 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.

21. 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.

22. P-- P-- P- P- P- P- G G G G G Cl- Cl- Cl- Cl- Cl- Cl- Cl- Cl- Cl- Cl-

23. P-- P-- P- P- P- P- G G G G G Cl- Cl- Cl- Cl- Cl- Cl- Cl- Cl- Cl- Cl-

24. P-- P-- P- P- P- P- G G G G G Cl- Cl- Cl- Cl- Cl- Cl- Cl- Cl- Cl- Cl-

25. P-- P-- P- P- P- P- G G G G G G G Cl- Cl- Cl- Cl- Cl- Cl- Cl- Cl- Cl-

26. 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

27. 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.

28. 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.

29. H2
pH 6.8, neutral
pH 8.8, negatively charged

30. P-- P-- P- P- P- P- G G G G G G G G- G- G- G- Cl- Cl- Cl- Cl- Cl- Cl-

31. P-- P-- P- P- P- P- G G G G G G G G G G G- G- G- G- G- G- Cl- Cl- Cl-

32. Myosin, 202 kd
MWs adjusted to account for masses of covalently bound dyes
-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