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The antibody that we did the IP with is not against USP25, rather a different protein. We identified USP25 via mass spec of a protein band from a SDS-PAGE analysis post-IP. Unfortunately there are very few USP25 antibodies available, yours was the first we tested. As I mentioned earlier we also did a western blot on Jurkat whole cell extract. Our data indicates USP25 mRNA to be expressed in this cell line at levels consistent with detectable USP25 protein expression.
Asked on May 06 2004
Many thanks for the additional information. I think we now understand your experiment and are almost convinced that you have shown that this Fast-Track antibody does not work. However given the lack of published work on USP25, especially work using antibodies, we want to ask one final question. What apparent MW was the USP25 band found at in SDS-PAGE? We just want to be reassured that the USP25 you observe isn't an alternatively spliced or proteolytically cleaved form that doesn't contain the RIMLSLSRTPADGR epitope that we used in manufacturing this antibody.
Answered on May 10 2004