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Immunprecipitation of phosphorylated proteins - (Mar/08/2006 )

i'm trying to immunoprecipitate this protein that is phosphorylated, from human tissue homogenate.
Just in fear of losing the phosphorylation, i have been doing homogenization, measuring protein concentration, immunprecipitation as well as SDS-PAGE and the transfer all in one day, which makes the day very very long!!! (when i need to do this experiment, i'm in the lab till really late).

Am i just being too paranoid? Anyone know what step would be the best to stop and come back the next day to continue, without risking the loss of phosphorylation on my protein?



do you use a buffer with something like orthovanadate?

when we are looking at phosphorylation state, immediately upon making the protein prep it is aliquoted and then flash-frozen with an alcohol/dry ice bath and put in -80; one aliquot is left on ice and quantified immediately.

Then, we can stop...and set up the western within a few days on the samples from the freezer.