Protein stability in RIPA buffer at room temp
Posted 13 July 2009 - 11:05 PM
I accidently left my RIPA buffer cell lysates in my ice bucket overnight. I came in the next day and there they were floating around in a pool of water. The tubes were sealed tightly but the samples hadn't been frozen for probably 12 hours.
Will I still be able to use the protein extracts for western blotting?
My RIPA buffer contains aprotinin, PMSF and B-mercaptoethanol.
Posted 14 July 2009 - 03:26 AM
Posted 14 July 2009 - 03:35 AM
What is worse : re-do the samples, or do western-blot for nothing ?
you could have a try, but if you have negative results you won't be able to conclude.
If you are looking for phosphorylation, I would be worry.
If you are looking for protein expression... depend on the stability of your protein of interest, but I would do the WB just to see. Of course you will have to repeat the experiment in anyway.
By the way, I always perform the experiment until the end when I do it for the first time, even I have more chance to have nothing, just to see if there are other troubleshoots to overcome (unless if the experiment is very expensive of course !)
Posted 22 July 2009 - 01:40 PM
That said, if your protein is inherently unstable at room temperature you may have degradation due to the protein just plain falling apart. So it basically comes down to a judgment call on your part about which step takes longer (collecting lysate or running western blot) and the costs associated with each. In my opinion, I say go ahead and run the lysate and see what you get.