Lambda Phosphatase Treatment of Cell Lysate - (Nov/11/2013 )
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?
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.cellsignal.com/products/images/2845_wb_fig1_ka_050518.jpg