Hi everyone,
Currently I mixed equal amounts of cell from SILAC experiment and freeze in -80. I'm planning to follow general protocol with double digestion (LysC+trypsin) and IP with anti-acetyllyssine antobodies. However I'm confused about inhibitors. In my global phosphoproteome I used protease and phosphatases inhibitors but for acetylome I don;t see any standardize procedure.
Could you please share some experience with me if I should use some cocktail of NIC and TSA or sth.
Thank you in advance.
Piotr
if u r using roch cocktail for protease and phosphatases. then no need to use anything else. go ahead
Hi there,
I disagree with Hazir Rahman and here is why: addition of protease inhibitors of general use (anti-trypsin like, anti-Me-pdependent protease etc) only prevent or slows down a site-specific protein degradation. But you are looking for Acetylated proteins specifically. Therefore you should add inhibitors of deacetylases into your Lysis buffer. Those are: sodium butyrate, Trichostatin A (TSA) and others (there is a huge list of them all with different specificities and and IC50). Usually two or 3 inhibitors are added (most often sodium butyrate ~10 mM final and TSA (final concentration may very)).
Good luck.
Its me again...Sorry for somewhat confusing message as you asked for PROTEASE inhibitors specific for acetylome experiments and I went on with all that info about HDAC inhibitors. Not that it is completely useless in this case as you need as much Ac marks preservation as possible but it was not what you asked for. My bad... it has been a very long day in the lab.
Good luck
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