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Phospho-protein vs total protein Western blot

phosphorylation PTM troubleshooting western blot

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#1 teechap

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Posted 03 September 2012 - 09:51 PM

So I'm running a pretty typical SDS-PAGE setup. Discontinuous gel (20% resolving), fresh sample buffer with BME/SDS, 90 degrees for 10 min before loading, etc. I'm probing with an antibody that recognizes a phospho-serine on my protein of interest, and I'm trying to normalize to the total amount of that protein after stripping and reprobing. The phospho-antibody is polyclonal and the total protein antibody is monoclonal.

The problem: my phospho-serine antibody gives bands exactly where I'd expect (~25 kDa), but the total protein band is all the way up at 250 kDa (no bands at 25 kDa). The only way I know how to explain this is I might need more BME to get rid of disulfide bonds, more SDS, or maybe a different pH in either the stacking or resolving gels. I've already tried not heating before I load, and I've made fresh sample buffer. Any ideas? I'm really stuck here!

#2 bob1

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Posted 04 September 2012 - 01:17 AM

Sounds like a non-specific band with your antibody rather than actually detecting the protein of interest. Try titrating the total protein antibody and see what happens.

#3 teechap

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Posted 04 September 2012 - 02:00 AM

I'm not sure what you mean by "titrating" the primary total antibody. If you mean just using different concentrations and seeing if I pick up a band at 25 kDa, I'm not sure this would help. I'm already probing 1:500 with the primary antibody. And it's monoclonal; it's been verified in many previous studies at ~25 kDa. The only difference is those studies used immune cells and I'm working with brain tissue....

#4 bob1

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Posted 04 September 2012 - 01:49 PM

Different tissue, different person working with it, all can easily lead to not finding the band at the appropriate size. If you are finding the phospho band at the correct size - then you are denaturing and reducing your protein enough that the non-phosphorylated form should also run at or about the same size... hence I think this is an antibody problem. Just because it is monoclonal, doesn't mean that it won't bind to other proteins non-specifically! Brain tissue is quite likely to have a different level of expression of the protein to other tissues. You can check out gene expression patterns for many genes at MOPED and BioGPS.

Yes, titrating means using different concentrations of the antibody. For this you will need to to probe the same sample run on different lanes in the same gel with the different concentrations. Since you are using 1:500 now, try 1:100. 1:200, 1:300,1:400 and see how you go.

#5 mdfenko

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Posted 06 September 2012 - 11:08 AM

you may be detecting an aggregate of your protein of interest (even in sds/bme proteins may aggregate when incubated for long periods at near boiling temperatures).

try reducing the sample incubation time at 90C to no more than 5 minutes or reduce the incubation temperature to 60-70C and incubate for 10-20 minutes.
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#6 teechap

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Posted 06 September 2012 - 02:20 PM

Thanks for the input! I'm trying all of this now...





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