7 replies to this topic

[sansub]

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

[sansub]

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

[Inmost sun]

sansub, on 18 June 2011 - 07:03 PM, said:

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

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

[sansub]

Inmost sun, on 19 June 2011 - 03:04 AM, said:

sansub, on 18 June 2011 - 07:03 PM, said:

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

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

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?

[bob1]

It will be fine to do that after storage.

[sansub]

bob1, on 19 June 2011 - 04:44 PM, said:

It will be fine to do that after storage.

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

[sansub]

What do you think I should do?

[bob1]

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