2D instead of Immunoprecipitation? - (Aug/24/2006 )
I want to sequence a band found in western blot, but the antibody didn't work in ip, can i run 2D gel, do transfer, then do IB to localize this band, and repeat 2D using exactly same condition and cut out the band of traget?
Does anyone has any experience of that?
I have once done a western from a 2D-Gel, worked o.k., my protein was detected in the western, but when I tried to cut it out for MS (out of the blotted gel), as imagined, there was to little protein left to be detected. I got results for another kind of protein with the same size and isoelectric point, must have overlaid my protein.
But you can try from a second gel, which you don't blot. Perhaps, if you have enough of you protein in the sample or have purified protein, then it could work. Give it a try.
Do you know the PI of your proein before run 2D, or you seperate protein with whole range PI gel first?
Sorry to ask this question, i am not familiar with 2D gel running.
Biomaus has given the right idea: Do two 2D gels run in parallel, blot one & detect your protein by the antibody, the other 2D-gel should be stained by CBB or silver; scan blot & stained gel and overlay them to detect the polypeptide to cut; 1 pmol protein is necessary;