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