general protein purification from mouse tissue - (Nov/23/2008 )

HI

what is the recomnded lysis buffer to purify all cytoplasmic proteins from mouse tissue (like : liver, kidney, testis, muscle etc.)?

I lysis the tissues with RIPA buffer but I did not get good purification (couldn raraly detect actin as a control)

Does someone have any idea????

thank you

Did you add protease inhibitors? If you didn't, it isn't surprising that you can't detect anything. Also make sure that your protein solutions are stored below -20 degrees C (-80 preferably) otherwise they will degrade very rapidly

RIPA buffer with protease inhibitors (cOmplete protease inhibitor from Roche is good) should work fine for the majority of soluble proteins. There are also some proteins that are largely insoluble - the methods section of this site should be able to help you there.

As far as I know RIPA always contains SDS.

1:25 for complete depends on how it was made up, but that is the usual dilution if you make a 25x stock - only lasts a couple of days-a week in the fridge, so store it frozen in aliquots is best.

The detergent in RIPA may solubilize membrane proteins, which you aren't interested in. Best not to have detergent unless you are after a membrane protein. My guess is that you don't have great lysis to begin with. I'd recommend cutting up the tissues and using a vigorous homogenizer such as a polytron, spinning down the tissues and re-polytronning the pellet several times. The supers should contain what you need. My buffer choice would be 50mM Tris, pH 8.0, 300mM NaCl, 20% Glycerol, + prot. inh.