| | Re: Western Blotting for Phosphorylated Proteins
I think it is essential that you add BOILING Laemmeli buffer to the crude sample, and that you boil immediately (not just the next day). Otherwise you may inadvertently facilitate proteolysis and/or dephosphorylation by providing an unfolded substrate.
If, say, there is a relatively stable protease in your crude extract it may have a 'field day' with an SDS-unfolded substrate until it too eventually becomes inactivated by the SDS. But it may have sufficient time to do a lot of damage. You need to make sure that inactivation is, as far as possible, instantaneous.
Phosphorylated alcohols such as phosphoserine (and 2,3-bisphosphoglycerate) are very stable in acid, so you could precipitate with TCA before adding Laemmli buffer. (A phosphorylated acid, or acyl anhydride, however, is not stable in acid).
Finally, I prefer to store samples containing SDS at minus 80 degrees C, rather than minus 20 degrees C. I find that MW standards in sample buffer deteriorate over time at minus 20C, for example.
Personally, I think a key consideration is to ensure inactivation is instantaneous. Cold quenching is another possibility