We have found that when we boil our sample for 2 minutes in SDS PAGE sample buffer we can detect phosphorylated forms of our protein of interest. But when boiled longer (5 minutes or more), we collapse those forms into a single band that is slightly larger in size compared to the non-phosphorylated protein. Has anyone experienced this? I would think phosphatases would be inactivated in sample buffer at high temperature so it is unclear why an even longer incubation should cause dephosphorylation. Unless it is a chemical reaction causing the dephosphorylation.
Michael,
you may experience beta-eliminations of your phosphorylated proteins.
As reference, I would recommend this one:
http://www.ncbi.nlm.nih.gov/pubmed/14622963
If you want to test whether this is happening in your sample, you just need to add a Michael donor and check whether this has been added to your protein (either by mass or if you use a fluorescent Michael donor, you can just check for fluorescence). Whether or not this can be visualized on a gel, is difficult to say. If you use a very high resolving gel, you may be able to check this by SDS-PAGE as well.
It is my experience that you do not and should not boil Phospho proteins or membrane proteins. Instead of boiling, incubate at 65C for 10 minutes. This eliminates aggregates and de-phosphorylation. I would also add a protease inhibitor cocktail, sodium fluoride (200 mM), sodium orthovanadate (200 mM), and phenylmethanesulfonylfluoride (200 mM)
I have been struggling with immunoblotting of phospho proteins as well, and during a master defense I also heard that it was important to not boil your samples at 99 but at 70oC. Is there any literature, Chad, that discusses this? I will definitely try it.
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