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Lambda Phosphatase Treatment of Cell Lysate

Phosphorylation Labmda Phosphatase Cell Lysate

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#1 Joe KooL

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Posted 11 November 2013 - 05:58 PM

HI,

 

I believe the protein encoded for in my cloned plasmid is phosphorylated. I've read that we can treat this protein with lambda phosphatase. the gene of this protein is cloned in a mammalian expression vector. Usually i'd transfect this plasmid into a mammalian cell line, let it incubate for 20 hours and directly harvest the cell lysate in laemlli buffer for western blot analysis.

 

How do i lyse the cells such that i can proceed with phosphatase treatment? 

 

 

Joe



#2 doxorubicin

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Posted 12 November 2013 - 02:07 PM

I used to do this frequently using NEB #P0753 (10,000U/ml for 1 hour).  Just scrape adherent cells in 1X Lambda PPase buffer, then sonicate briefly, add the Mn and phosphatase and incubate at 37C for one hour.  My results looked like this:  http://www.cellsigna...1_ka_050518.jpg







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