2 replies to this topic

[microRNAboy]

Hi All,

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!

[madrius1]

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!

[microRNAboy]

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!