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SDS-PAGE

SDS-PAGE. 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. HRP. + ECL substrate  light. HRP conjugated 2 o antibody. 1 o antibody.

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SDS-PAGE

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  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 2oantibody 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-PAGEpreparation 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 mMglycine, 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. G G G G G P-- P- P- P- Cl- P- Cl- P-- 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-

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

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

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

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

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

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