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BATCH NUMBER 90293 ORDER NUMBER 147797 DESCRIPTION OF THE PROBLEM no bands SAMPLE STC-1 cells (mouse intestinal endocrine) lysates PRIMARY ANTIBODY abcam rabbit anti-PC1(ab12275) or abcam rabbit anti-PC2(ab3532), 1:2500 or 1:500, 0.01%tween-20/PBS, 1hr RT or overnight 4C plus 1hr RT washing: 2x 1min, 1x 15min, 3x 5 min DETECTION METHOD ECL Plus (Amersham, cat RPN2132) POSITIVE AND NEGATIVE CONTROLS USED loaded STC-1 lysates ANTIBODY STORAGE CONDITIONS antibody dispensed in 2uL aliquots and stored at -20C SAMPLE PREPARATION lysate buffer: complete mini protease inhibitor (Roche, cat #11836153001) AMOUNT OF PROTEIN LOADED tried 10 and 20ug of total protein lysates per lane ELECTROPHORESIS/GEL CONDITIONS 95C at 5 min, DTT, NuPAGE 4-12% Bis-Tris gel (Invitrogen), 150V 1.5 hrs TRANSFER AND BLOCKING CONDITIONS 10% methanol/NuPAGE transfer buffer, 30 V 1.5 hrs, blocking: 5% skim milk/PBS/ 0.01% tween-20 SECONDARY ANTIBODY goat anti-rabbit HRP conjugated (another company), 1:10,000, diluted in 0.01% tween-20/PBS washing: 2x 1min, 1x 15min, 3x 5 min HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 3 HAVE YOU RUN A "NO PRIMARY" CONTROL? No DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes WHAT STEPS HAVE YOU ALTERED? 1) increased primary antibody conc from 1:2500 to 1:500 2) increased incubation time from 1hr RT to overnight at 4c plus 1hr RT 3) increased secondary antibody conc from 1:10,000 to 1:5,000 ADDITIONAL NOTES To test whether any protein was transferred, I probed the same membrane that did not yield any bands with the anti-PC1/PC2 antibodies, with anti-B tubulin and then HRP conjugated secondary. Using this antibody I was able to detect the appropriate bands for this ubiquitous protein.
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ANSWER: |
Thank you very much for your protocol details and your order number. I firstly looked at your protocol and think it is very good. I found references for the presence of both PC1 and PC2 in STC-1 cells (Yoon et al, Endocrinol. 1997 for example) and therefore the problem is certainly not due to low amounts of the protein in your samples. However, we have not determined whether ab12275 does cross react with mouse PC2 and I am currently enquiring to find out the exact homology of the immunogen with the mouse protein; I performed a homology search with the last 15 amino acids which contain the immunogen and found 86% homology. I think therefore that it would be important to determine the exact homology so that we can determine if the problem with ab12275 is a species cross reactivity problem. I secondly looked at the shipping records of your order and indeed found a delay; the package was held by regulatory agency on May 24th and I am worried that the package was stored in a hot area. I would like to offer you a replacement vial of ab3532 already and to wait for the cross reactivity information regarding ab12275, as if it is unlikely to cross react with mouse I would recommend to use a different antibody to detect PC2. I appreciate your patience and will e-mail you as soon as I find out the missing information regarding ab12275, |
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