Two bands in WB but one band in IP - (Apr/15/2015 )

Hi,

My protein of interest is around 70 KD and I have seen there is another isoform at 100 kD. When I do western I can see  both the 70 and 100 kd band. However, in my Immunprecipitation I could never see the 100 KD band , only the 70  KD band pulls down with my IP. This I have seen more than 20 times , so its reproducible.

I was wondering, what might be the possible explanation for it. Why the 100 KD disappears in IP ? any good scientific explanation will be highly appreciated.

Thanks.

are you using the same antibody for both?

the isoform at 100 kDa may have the recognized epitope hidden when not denatured.

Hi,

try to perform IP using denaturated conditions

http://www.cellsignal.com/contents/resources-protocols/immunoprecipitation-protocol-(for-denatured-proteins)-(ip-denatured-protein)/ip-denatured/