protein sample should be intact in -70c as far as I know.
but when I stored them 1 week after first western blot, I can't even detect b-actin signal in the second experiment.
I used cold RIPA buffer directly in the cell culture dish with protease inhibitor cocktail
and centrifuged 13000rpm 5min.
does 5min centrifuge would be the problem?
please reply if anyone knows the reason or has a experience.
thank you!
Hi Kyutae,
I cannot comment on the expression of your protein, but can say that proteins are indeed stable at -70C. Problems can arise during the freezing and thawing processes when proteins can concentrate and aggregate. Snap freezing can help minimize issues, but may not be enough for your protein. You can try formulating the solution with something like trehalose. I've attached a review article on formulation that can give a more complete picture, I'm definitely not a formulation expert. Good luck!
How were your total protein assay measurements? Is the yield fine for performing a western? Is the SDS-PAGE monitoring OK? Have you checked the transfer using Ponceau S? If these are OK, and assuming an identical procedure, freshly prepared buffers have been applied, and your antibody is still working for b-actin....
Centrifugation and cold storage should not be a problem if the proteins are not aggregated over the time course during cold storage and hence remain in the inclusion body pellet. To clarify this, increase the RIPA buffer concentration or use different/additional solubilization agents for the previously lysed cell extract to investigate if the b-actin and your target protein are in the pellet.
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