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BATCH NUMBER 119201 ORDER NUMBER C0144771 DESCRIPTION OF THE PROBLEM Wrong band size, multiple bands SAMPLE Cell extract PRIMARY ANTIBODY ab17008 (abcam) DETECTION METHOD ECL POSITIVE AND NEGATIVE CONTROLS USED Pos. control: Lentiviral overexpression ANTIBODY STORAGE CONDITIONS Aliquots stored at -20 SAMPLE PREPARATION Protease inhibitor cocktail, heating, reducing AMOUNT OF PROTEIN LOADED 15 ug ELECTROPHORESIS/GEL CONDITIONS Reducing TRANSFER AND BLOCKING CONDITIONS According to ECL protocol (Amersham) SECONDARY ANTIBODY antiGoat HRP BioRad HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 2 HAVE YOU RUN A "NO PRIMARY" CONTROL? Yes DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes WHAT STEPS HAVE YOU ALTERED? None ADDITIONAL NOTES Scan of Western submitted in previous e-mail
Asked on Aug 23 2005
Thank you for taking the time to send us your protocol details, this was very useful for me to understand your problem. We have not tested this antibody in the same samples as yours; in our hands, our positive control of human heart lysate gave a single band at the expect MW of 41kDa. Could you please clarify if you have tested in WB your samples for KLF2 before? I am wondering if the samples express a different form than the protein found in human heart lysate samples. Could you also tell me a little bit more about your blocking conditions: what you blocking with, for how long and if you added the blocking agent to the antibody dilution buffer. Sometimes blocking can prevent adequate binding of the antibody to the membrane and create non specific binding and/or no binding to the band of interest. We typically recommend a 1hr blocking step with 5%BSA or 5%milk in TBST and incubate the membrane with the primary and secondary antibodies diluted in TBST only. Please be assured that under the Abcam guarantee if you run our positive control in WB and find similar problems we will offer a replacement or refund if the antibody was purchased in the last 90days. I was unable to find an order with the reference C0144771, did you purchase the antibody through one of our distributors? This is not a problem if this is the case, we can liase with the distributor to arrange a refund or replacement if you find that the human heart lysate samples give problems too. I hope the above information will be useful, if you require further assistance please not hesitate to contact me,
Answered on Aug 23 2005