Preparing cell lysate from cell culture for Western blot - (Oct/16/2007 )
I was wondering if anyone could direct me to a good protocol for how to prepare a cell lysate from a cell culture for use with SDS-PAGE (Western blot).
wash cells with PBS
add 1x SDS buffer (with protease inhibitor)
Collect the extracts, sonicate and store -80C
please find the attachment for the LYSIS buffer composition
-wash cells with ice cold pbs.
-lyse cells with appropriate amounts of lysis buffer.
-collect the lysate with cell scraper.
-centrifuge at 14k/10/4C.
-collect the supernatant in fresh tubes.
-estimate protein concnetration with bradford.
-load the gel.
Full protocol is attached.