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Protein stability in RIPA buffer at room temp


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#1 labgrl76

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Posted 13 July 2009 - 11:05 PM

Hi,

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.

Thanks

#2 neuron

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Posted 14 July 2009 - 03:26 AM

Before going for western blotting, why don't you check your protein on gel. Your protein might have degraded by now, so directly western blotting without checking the protein, will not be a good idea.

#3 little mouse

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Posted 14 July 2009 - 03:35 AM

I don't know your samples.
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 !)

#4 polyfractal

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Posted 22 July 2009 - 01:40 PM

As little mouse mentioned, phosphorylation of proteins will likely be vastly decreased due to free phosphatases. Quality of the protein will likely depend on the inherent stability of the peptide. While PMSF degrades rapidly in water (half life of about 100 minutes) it does, bind permanently and proteases that are affected will be inactivated permanently. The B-mercapto should also help remove proteases from the picture due to breaking up disulfide bonds. Degradation due to proteases will likely be minimal.

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.




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