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Preparation of mammalian cell lysis


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7 replies to this topic

#1 sansub

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Posted 18 June 2011 - 06:37 PM

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

#2 sansub

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Posted 18 June 2011 - 07:03 PM

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

#3 Inmost sun

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Posted 19 June 2011 - 03:04 AM

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

#4 sansub

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Posted 19 June 2011 - 10:04 AM


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?

#5 bob1

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Posted 19 June 2011 - 04:44 PM

It will be fine to do that after storage.

#6 sansub

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Posted 19 June 2011 - 06:27 PM

It will be fine to do that after storage.



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

#7 sansub

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Posted 19 June 2011 - 06:36 PM

What do you think I should do?

#8 bob1

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Posted 20 June 2011 - 04:55 PM

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




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