When preparing samples for loading on Western blot, I normally add the appropriate amount of loading buffer (LDS), and heat the samples (e.g., 5 min. at 94C). Is this okay to do for samples when I am trying to detect phospho-protein (phosphorylated protein)? Thanks for any tips.
hi Corey
usually yes, it is more important to use appropriate amounts of phophatase blockers and protease blockers to get consistent results.
best, Chris
Hy Corey,
indeed, works just fine. as Chris pointed out, use blockers, be fast and work on ice.
Best
Christian
As you are using LDS sample buffer, I am assuming you are doing denaturing gel electrophoresis. In that case, it is required to heat the sample. So, you are fine. But for non-denaturing or native gel electrophoresis, make sure you don't use buffer that contains SDS and don't heat the sample.
Hope it helps.
Thanks
My answer is yes but it could depend on the phosphoprotein or peptide. We do it for PKB and MAP kinase. I would not heat for more than 1 min at 95 °C
Hi Corey! Yes, it would be ok to boil your ssmples right before loading them on the gel, for immunoblotting of phospho-proteins.
Additional tip: Use protease and phophatse inhibitors in the lysis buffer to make your lysates..
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