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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

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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.

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The human genome has 518 protein kinases

Phosphorylated proteins mediate: cell division (cancer) cellular differentiation hormonal signal transduction

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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.

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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),

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P-threonine and P-serine antibodies are weak and expensive!

Company Antigen Dilution Cost/2Dblot

Abcam phospho-serine 1:500 $330

Abcam phospho-threonine 1:200 $330

Typical mouse monoclonal antibodies for Western blotting

Many companies sell these abs.

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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)

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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.

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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.

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Tries 1-9 with 1D optimization using rat liver homogenate are not shown.

Try 10, 2DE, next slide…..

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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.

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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.

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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.