Lysis buffer - (Jan/25/2006 )
I'd like to know if someone could tell me about lysis buffer storage conditions... I mean, I prepare it using:
Tris 50 mM
NaCl 150 mM
EDTA 1 mM
PMSF 0.1 mM
beta-glycerol phosphate 100 uM
Triton X-100 1%
The question is... Could I prepare this buffer and keep it at -20ºC until use or some component would be degraded if it is frozen??
I'm not sure your application, but this looks very similar to our buffer for lysis of tissue cells, in order to make cell extracts for westerns looking at phosphorylation state of proteins....
What works for us, is to make the lysis buffer and store in aliquots at either -20 or at 4. The kicker is, don't add the protease inhibitor or the orthovanadate until immediately before use, and then only thaw what you need, add the additives, and toss anything you don't use.
I don't actually have experience with the orthovanadate or protease inhibitors degrading, we just have always done it like this 'in case'
does this help?
The PMSF will degrade.
Prepare the protease inhibitor fresh...the other stuff should be O.K. (if I'm reading it right).
Thank you for your help!!!