Western Blotting for Phosphorylated Proteins

[tgd]

Hello,

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

Good luck,

TGD

[admin]

Also when i was doing my primary antibody incubations on the phosphorylated protein I was doing them overnight in the cold room as the signal was weak.

[snoopygirl]

You might also try changing your wash buffer to a TBS + 0.1% Tween-20. Using PBS might be adding to your background as there are phosphates in your buffer.

[msneve]

I am just beginning to work with a phosphoprotein antibody. One item which I have read says to avoid milk based blocking solutions because they contain many phosphorylated proteins which will contribute to high backgrounds and quench your antibody making detection of your specific bands difficult.

[Fonzy99]

U should use NaF and Vanadate in addition to your complete protease inhibitor cocktail

[5µl-pipette_death_punch]

Hi guys!

I would like to continue this thread. I deal with the same problems as sciencemandan. I'm currently after the phosphorylated version of TrkB receptor. As TrkB is an integral membrane protein I applied an specialised lysis buffer for membrane proteins, which shows good results with normal TrkB. This buffer contained a pptase inhibitor mix, but I didn't obtained any bands, whereas the control with TrkB worked perfectly. So how long do you guys use these inhibitors? Theoretically after the SDS-page the thread of pptases should be eliminated?

Many thanks in advance.

best wishes from cold Insbruck...

[gehrs]

the PhosStop from Roche is really good.

A cheaper approach should be the 2 solutions from sigma.aldrich.

and ever cheaper sodium fluoride ad sodium orthovanidate (1mM)