Samples for Western to detect phosphorylation of EGFR - (Nov/11/2010 )
I'm really new to cell-signalling and am looking for some advice regarding preparation of my samples that I will be using in a Western to detect phosphorylation of EGFR.
I have been lysing my cells in the culture dish in ice-cold RIPA containing protease and phosphatase inhibitors, but am having trouble lysing in a small enough volume to then load 50ug protein onto my SDS-PAGE gel.
Is it common to scrape cells into ice-cold PBS, pellet and the resuspend in a small volume of lysis buffer? I'm worried that the transient EGFR phosphorylation will be lost the longer the samples are handled before lysis.
Any advice greatly appreciated!
Keeping the cell lysate on ice greatly reduces the loss of phosphorylation. Also, I would recommend not to freeze the cell extracts before blotting, as a strong decrease in protein phosphorylation is often seen upon freezing/thawing. Do you add phosphatase inhibitors to your cocktail, such as sodium vanadate and sodium fluoride?
I routinely scrape cells in PBS before lysis. Works like a charm!
Thanks for your reply!
Yes, I add sodium orthovanadate and sodium fluoride to my RIPA buffer.
That's set my mind at rest. I'll scrape into ice-cold PBS before lysis! I had noticed a big difference in my results when comparing fresh and freeze/thawed samples. I'll use fresh lysates from now on too!