Problems with cell lysate - (Mar/15/2006 )

Hi,

I'm trying to lyse approximately 7.5million 293T cells using 200ul of RIPA buffer (which also contains 2X protease inhibitor), but the lysate resulted is very gluey. This RIPA buffer was made last week and I used it to lyse other cells, which were perfectly fine on a western blot. What do you think went wrong? Could it be that the RIPA buffer/protease inhibitor has degraded over a week (it was stored at -20C, thrawed once since I last used it)? Any suggestion will be greatly appreciated. Thanks!

You might want to increase the amount of Ripa for lysing 7.5 million cells. This is kinda the problem I had while using RIPA and I thought that the problem was to do witht he SDS in the buffer. I have heard that SDS precipitates out at very low temps ( i dont know if this is true) ...Anyway my problems went away when I switched to a Non-SDS based cell lysis buffer.. Also my protein yeild is better. I dont think this has anything to do with the protease inhibbitors.

good luck

lemme know if you need the recepie for cell lysis buffer.

hi,
i do feel that u r using low volumes of lysis buffer.
gud luck

Hi yall

Thanks a lot for the kind replies! I increased the buffer volume and spinned for 45mins, eventually all the gluey stuffs settle at the bottom nicely.

Thanks all!