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detection of phosphorylation from cell pellet


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#1 soymilk14

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Posted 20 March 2012 - 11:50 PM

Hi guys,

I want to collect my cells treated with different concentrations of drugs and detect its phospho AKT and Phospho ERK. I however can not run SDS immediately. I heard that detection of phospho level should be done immediately after harvesting the cell pellet.
My question is: which is better, can i keep the cell pellet at -80 degrees and lyse the cell next time? or keep the cell lysate at -80 and do western blot next time? can i still detect the phospho level by doing this?

I hope you can give me tips on how to keep the cell stock or cell lysate prior to western blot. Thank you.

#2 mdfenko

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Posted 21 March 2012 - 07:39 AM

if you keep protease and phosphatase inhibitors in your lysing buffer then i would lyse and store. but it would be best to use the lysate immediately.

you could (should) do an experiment where you lyse and aliquot. then use one aliquot immediately and freeze the rest. take an aliquot at fixed periods and determine if the phosphorylation status varies from the initial.
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#3 Inmost sun

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Posted 01 April 2012 - 10:28 AM

as mdfenko says, use phosphatase and protease inhibitors; try to shock freeze lysates, which is to recommend before storing at -80°C




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