Preparation of mammalian cell lysis - Preparation of mammalian cell lysis for Western blotting (Jun/18/2011 )

How do I make cell lysates for mammalian cell lines- NIH-3T3 fibroblasts using protease inhibitor? Can someone give me the protocol?
Thanks!!

What if I do not centrifuge the cells after adding the protease inhibitor and cell lysis buffer to them? Please help!

sansub on Sun Jun 19 03:03:11 2011 said:

then you have full lysate with organelle debris, DNA and cytoskeletons; you may get problems with dissolving this kind of lysate with SDS sample buffer

Inmost sun on Sun Jun 19 11:04:08 2011 said:

Thank you for your reply. Well, I did not centrifuge my samples and simply stored them at -20 degrees. Do you think it is possible to centrifuge it after thawing the sample. Would I be able to get rid of the debris this way?

It will be fine to do that after storage.

bob1 on Mon Jun 20 00:44:24 2011 said:

I tried doing that. But, I did not see any pellet

What do you think I should do?

Try running the lysates on a gel. If you can see the protein after staining, they will be fine.