I have created several stable cells which express shRNAs to downregulate BRCA1 in HeLa cells. I have extracted protein in RIPA buffer (with sodium vandate, PMSF and protease inhibitor) and run this on an SDS-PAGE and then transferred onto a PVDF membrane.
I have used the Ab-1 BRCA1 antibody from Calbiochem which we have used successfully in our lab before (the epiptope is within the N terminus of BRCA1).
I get a beautiful doublet band where BRCA1 should be (double because BRCA1 has a phosphorylated form) which is decreased in my different shRNA constructs.
Beautiful I thought! Its worked!!
Then my supervisor had a look at it and noticed that a band just under the 150 kDa mark which I thought was non specific binding but she thinks may be the delta 11 BRCA1 isoform which is published to be 110 kDa.
Unfortunately, as most people opt to only show a tiny window of their WB bands to show results, I can't tell whether other people who have published BRCA1 knockdown, also got this band or found knockdown of the delta11 isoform.
Has anyone used this antibody for BRCA1 WB and/or BRCA1 shRNA and also had this same problem?
Edited by Ania_w, 30 April 2012 - 03:17 PM.