Cell Lysates from a Western Blog - (Mar/24/2011 )

Hi All,

Today - I made a silly error. I had 3 wells that I needed to get cell lysates from (from a 6 well dish). I washed the cells with ice cold PBS, added the NP-40 lysis buffer, and then scraped it quickly thereafter (but not on ice). We don't usually incubate the cells on ice after adding lysis buffer before scraping because it works just fine. I added the cells to tubes and then incubated that on ice for 20 min.

I'm afraid that my protein degraded while it was sitting in lysis buffer at room temp for a few minutes. Usually, I scrape on ice, but just forgot to today. Do you think this is a possibility, or does it take longer for proteins to degrade?

Thanks.

Did you have protease inhibitors in the lysis buffer? If so, I think they will be OK, though how long is a few minutes (10, 15, 30...)?

bob1 on Fri Mar 25 00:08:36 2011 said:

Yes, I had protease inhibitors in the buffer. I would say no more than 10 minutes (I added the lysis buffer, scraped, put in tube on ice) for 3 small wells.