How to do the lysis of cells without white stuff? - (Mar/19/2007 )
I work with Hela, HEK and COS cells. For lysis I use RIPA buffer with vanadiate sodium and inhibitor of protease. The problem is, when I want to do the Bradford assay (to measure the concentration of protein), after pipetting, some white stuff mostly occurs, so I can't even take required amount of liquid... supposingly it's DNA and other products of cell lysis..
So, I tried to improve the lysis, and after adding RIPA, I pipetted it about 30-40 times till I couldn't see any floating pellet (It looks like a clear solution) I froze it in -80C for 5-10 minutes and then thaw it on ice.. .surprisingly I couldn't see any white stuff but my sample became as a 'jelly'! I couldn't even take required volume to measure the concentration.. I vortex it, and then centrifuged in +4 for 2 minutes, and on the bottom appeared the white ‘jelly’ stuff, but I could take a liquid from the top to measure the concentration.. still it doesn't work well for me.. I tried to do the Bradford to have less than 5% difference between readings (I take 2ul to one tube and 5ul to another and I add 2 ml of Bradford reagent)...
I would like to ask for advice - maybe I did something wrong? does anybody know how to do that without this white stuff?
Thanks for the answer!
i am sorry i dont know if i got you right or not... you mean you dont centrifuge your cells after RIPA lysis?
i do my lysis with NP 40 with protease inhibitor and phosphatase inhibitor * HEK cells*but after 30 min i do centrifuge my cells for 15-20 minutes at 150000rpm 4 degree then i got clear lysate and a bellet i measure my protein in the supernantent.
hope this help