2 replies to this topic

[anemia]

I was told recently that it is not good to snap freeze the sample first and lyse the tissue later, because proteins might degrade during freezing and thawing processes. Is that saying reasonable? Can I get good Co-IP and Western blot results with brain samples that are snap-frozen first and homogenized later

Thank you.

Edited by anemia, 21 April 2009 - 10:56 AM.

[Dr Teeth]

anemia, on Apr 21 2009, 02:54 PM, said:

I was told recently that it is not good to snap freeze the sample first and lyse the tissue later, because proteins might degrade during freezing and thawing processes. Is that saying reasonable? Can I get good Co-IP and Western blot results with brain samples that are snap-frozen first and homogenized later

Thank you.

Yes.  Include protease inhibitors in your buffer and keep your samples cold at all times including thawing the lysates on ice, and you shouldn't have any problem.  It is funny, people say, "Oh, you will have degradation, etc." but these same people usually haven't tested this at all.  At any rate, with the transcription factors that I am interested  in, freezing has never been a problem.  Results duplicate those of non-frozen samples.

[anemia]

Thanks for your very helpful comments. Actually I always do like that (snap freeze and store the samples first) and I know many people do like that. The only thing I am not sure is whether there is some literature reports the difference between the two approaches. For snap freezing method, I could find many literature supports.

Edited by anemia, 21 April 2009 - 11:03 PM.