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BATCH NUMBER -- NOT SPECIFIED -- ORDER NUMBER -- NOT SPECIFIED -- DESCRIPTION OF THE PROBLEM multiple nonspecific bands with background. no specific bands revealed SAMPLE cell extracts from transient transfected 293T cells. E2-p300 fusion expression plasmids were transfected superfect was used as transfect reagents PRIMARY ANTIBODY E2 antibody (1:1000) ab1072, fresh shipment DETECTION METHOD ECL plus POSITIVE AND NEGATIVE CONTROLS USED positive control: 1) same cells were transfected with cyclin D1 and subsequent western blot was beautiful; 2)same cell lysate was bloted with another antibody, (anti-GDI), the results was nice and clean; our conclusion is that the E2 primary antibody does not work! ANTIBODY STORAGE CONDITIONS 4C SAMPLE PREPARATION standard tris buffer plus np-40, pmsf, dtt and protease inhibitors(tablet) AMOUNT OF PROTEIN LOADED 60ug ELECTROPHORESIS/GEL CONDITIONS 7% sds-page TRANSFER AND BLOCKING CONDITIONS semitry transfer, 5% milk in tpbs SECONDARY ANTIBODY anti-mouse-HRP conjugated HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 1 HAVE YOU RUN A "NO PRIMARY" CONTROL? No DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes ADDITIONAL NOTES We are well experienced lab with many publications each year,and western blot is a routine in our lab. we have applied positive controls(for transfection, primary antibody, and 2nd antibody) for the western blot. E2 antibody did not pick up any specific signal other than many nonspecific bands. please ship another antibody that DO works in your hands, or please refund us, so we could buy E2 antibody from other sources.We are addressing reviewer's question for a paper, please understand that we are underpressure and we need to get the E2 western blot to work within 2-3 weeks. thank you.
Asked on May 05 2005
I'm sorry to hear you are having problems with ab1072. I am currently trying to find out more information about the protocol used to test the antibody in WB. Have you tested the blot without milk blocking and if the non specific bands could be due to the secondary antibody? How long did you incubate the primary antibody for? The absence of a band could be due to the levels of Bovine Papilloma Virus E2 being below WB detection levels, I am trying to find a positive control to help you and I apologise for the delay and appreciate your patience,
Answered on May 10 2005