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Are proteases a problem in protein quantification assays? - (Jul/31/2010 )

Hi friends,
I'm becoming familiar with the BCA assay. I have tissue extract from 30 animals of which I need to quantify the individual protein content. Diluting each of these extracts to a proper concentration takes quite some time, so by the time the last one has been diluted the first samples has been thawed for like 20 minutes. Should I be worried about protein degradation during that process? I have no feeling for how much a concern proteases are in general. I might just be paranoid from spending too many hours in the RNA lab, where RNAses are quick to punish inconsiderate behavior ;)

-Jens Madsen-

why not use protease inhibitors? ever heard of them? PMSF? Roche cocktail tablets?
you always need to add them to your lysis buffer. I wouldn't dare keeping my protein extracts 20min at room temperature.


protease inhibitors may interfere with the assay. you can keep your diluted samples on ice until you add the bca reagent.


i always add a protease inhibitor (fresh) to my homogenization and/or solubilization buffer, depending on how i'm preparing my tissue samples, before the BCA assay; i always keep my protein on ice, except when i'm preparing the BCA assay itself - then i just take the 2 or 4ul of protein and dilute it at room temperature while proceeding with the assay.

i've never heard that protease inhibitors could interfere with the assay...