I have a doudt in adding protease inhibitor. Should i add the cocktail both in cell lysis buffer and column buffer to prevent my protein from degradation during purification and while storage? or should i add only in cell lysis buffer? what is the best temperature for long term protein storages?
Protease inhibitors are usually added only to the lysis buffer. If you add inhibitor according to protocol you pretty much knock out all proteases that are targeted by the inhibitor. Make sure you use the correct inhibitor cocktail depending on your purification protocol - don't use EDTA or only minimal amounts for Ni-NTA purification. If you don't use Ni-NTA you can add EDTA in all column buffers. EDTA is cheap and may add to protein stability. There are other components you can add to the column buffer to add to protein stability like reducing agents like DTT, glycerol or maintaining a certain molarity of your buffer or salt in the buffer. This is to maintain protein stability in an in vitro environment compared to protection from proteolytic degradation during lysis. The composition of your column buffers depend on your protein.
Protein samples are best stored at -80C for long term.
A note: Before actually choosing / adding the inhibitor cocktail check whether your protein is degrading. There some proteins, which are pretty resistant, some others are not. If you are lucky you may get away without inhibiting proteases. However if you need protease inhibition, the best to actually choose the best strategy compatible with your downstream separation proceudres and assays. This latter is not a trivial task, but worth to do.
Dear
You can read also this publication: Methods in Enzymology (1990) volume 182. A Guide to Protein Purification. Edited by M. P. Deutscher. Academic Press.
Hoping it will be helpful
I had to use protease inhibitors in both my lysis buffer and throughout purification to keep my proteins of interest from being degraded. As for long-term protein storage, if you need to perform measurements of enzymatic activity, in addition to storing at -80 deg C, you should consider adding an antifreeze such as 10-20% glycerol or sucrose to preserve the protein's stability.
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