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IP/Western complication


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#1 Russell

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Posted 01 July 2004 - 09:18 PM

There is evidence that 2 proteins that we are interested in become bound together upon the phosphorylation of 1 of the proteins. We would like to demonstrate the binding by IP'ing one of the proteins and blotting for the other protein. This procedure has been successful; however as luck would have it, both proteins are almost identical in size (92 vs. 95 kD), so it is difficult to completely convince others that the blot signal is indeed from the 2nd protein and not non-specific binding of the IP'ed protein. So what we have is an IP for protein #1, with separate blots for protein #2 and protein #1 (the latter done to show that the IP actually worked) that look basically identical. Any advice on how to show that these are truly 2 separate signals other that just assuming that the blotting antibodies worked specifically?

Thanks,

Russell

#2 phdconsult

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Posted 03 July 2004 - 04:14 PM

This is an extremely elegant problem.

I would firstly establish another line of evidence for complex formation using gel filtration where an additional peak should be visible at 187kDa after the phosphorylation event- which can be demonstrated with either antibody.

This complex of the two proteins P1-P2 can be physically separated on a 15% SDS-PAGE and you can actually see the doublet if certain run conditions are met. Here is how.

Cast a 15% denaturing gel with just the separating gel-no stacking.

This gel must have a run length of at least 10 cm. Mini gels are out.

Use prestained markers

Run the electrophoresis until the 100kDa size marker is in the middle of the gel.

Transfer and blot with- 1. P1+ P2 antibody combo; 2. P1 Ab alone; 3. P2 Ab alone 4. Ab to phosphoamino acid (Sigma) and 5. Ab to P1 + Ab to phosphoaminoacid combo.

Good Luck

#3 Sprag

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Posted 19 July 2004 - 02:27 AM

Alternatively, you could add a tag to one of the protein (i.e GFP/Myc/Flag). This would shift the molecular weight and therefore you would be able to separate them on a gel.

-sprag-




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