1. 1 Talk Outline  Excerpt from talk given Nov 06 at the American Electrophoresis Society meeting in San Francisco  P-Serine & P-Threonine Western blotting with ECL Advance from GE Healthcare

2. 2 2D gels contain TMI (too much information). How to simplify? Focus on subsets.  Subcellular fractions Mitochondria, nuclei, microsomes etc. But often the subcellular fractions are complex too.  Phosphorylated proteins using Western blotting Antibodies give an enormous increase in sensitivity, 10-100X silver staining.

3. 3 The human genome has 518 protein kinases Phosphorylated proteins mediate:  cell division (cancer)  cellular differentiation  hormonal signal transduction

4. 4 Western blot protocol 1.Perform 1D or 2DE on a sample prepared in the presence of protease and phosphatase inhibitors; transfer the proteins to PVDF. 2.Wet the blot in methanol, rinse, block in 5% BSA. 3. Incubate with anti-Phosphoprotein ab overnight. 4.Wash 3X. 5.Incubate the PVDF in secondary ab (goat anti-mouse HRP conjugated) for 2 hr with shaking. 6.Wash 3X. 7.Use GE Healthcare’s enhanced chemiluminescence reagent (ECL or ECL-Advance) to detect signal.

5. 5 P-Tyrosine Westerns work well, but we currently have more calls for P-Serine & P-Threonine Western blotting than P-Tyr. Approximately 30% of cellular proteins are phosphorylated, most at serine and threonine residues (Philip Cohen, TIBS 25, 596, 2000),

6. 6 P-threonine and P-serine antibodies are weak and expensive! CompanyAntigenDilutionCost/2Dblot Abcamphospho-serine1:500$330 Abcamphospho-threonine1:200$330 Typical mouse monoclonal antibodies for Western blotting Many companies sell these abs.

7. 7 Results have been poor. Typical results with ECL for 1:200 dilution antibodies are shown below. (Coomassie stained PVDF on left; ECL film on right)

8. 8 ECL Advance from GE Healthcare GE web site says ECL Advance sensitivity is pg, much greater than ECL, so primary ab concentrations may be reduced.

9. 9 We chose Qiagen antibodies: PhosphoSerine ab Q5 (IgG & IgM) for detection of phosphorylated serine residues, irrespective of surrounding amino acids PhosphoThreonine ab Q7 (IgG) for detection of phosphorylated threonine residues, irrespective of surrounding amino acids Both are $340 for 100 ul; suggested dilution is 1:200, enough for 1 2D gel.

10. 10 Tries 1-9 with 1D optimization using rat liver homogenate are not shown. Try 10, 2DE, next slide…..

11. 11 Qiagen Anti-PSer versus Anti-PThr for 2D gels from same rat liver homogenate These 2D patterns are excellent. Similarities between the patterns would be expected since it is known that the P-Ser & P-Thr kinases are not very selective.

12. 12 Anti-PSer + anti-PThr combined: The abs were combined before the dilutions. 1:4000 clearly gives a darker yet clean pattern. (Secondary ab was not changed.) This is what we’re currently using for clients.

13. 13 Summary  ECL-Advance gives a dramatic increase in sensitivity for P-Serine and P-Threonine Western blotting. It’s a major breakthrough.  We’re not sure of the specificity of the Qiagen antibodies. They don’t pick up casein for example. We can’t be positive that all proteins detected are phosphorylated.  Differences between control and test samples should be valid even if some nonspecific binding occurs.