Immunoprecipitation - Can I boil then freeze? - (Aug/28/2006 )
I am new to immunoprecipitating proteins. My protocol says to add sample buffer and boil the lysate after immunoprecipitating to release the bound proteins. However, I need to do a protein quantification assay before running the Western. Also, I would like to freeze the lysate and do Westerns with it at other times.
Does anyone know whether it is OK freeze the lysate after it has been boiled? Will this degrade the proteins significantly? Also, if I am able to freeze, do I need to boil again after thawing and before doing the Western?
Thanks in advance,
you should have no problem freezing and defrosting and using your samples. you should not have to reboil but be careful that the entire sample, especially the sds, is back in solution before using (you may have to warm slightly).
remember, standards are treated this way.
As mdfenko said, you shouldn't have problems in freezing and then thawing: I do it very often. Regarding your quantification, I find it better to quantify the proteins before the IP, rather than after, because you will have a lot of Ab, proteinA or G etc and this will not make the quantification easy or precise. You should rather start from the same amount of proteins