Question (18768) | Anti-Aurora B antibody [mAbcam 3609] (ab3609)

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Question

BATCH NUMBER 123472 ORDER NUMBER 20600471 DESCRIPTION OF THE PROBLEM No signal SAMPLE Human cell lines, CAHPV10, Hela, Du145, whole protein extract. PRIMARY ANTIBODY ab3609, 1 hr in 0.5% milk DETECTION METHOD ECL POSITIVE AND NEGATIVE CONTROLS USED ab9484 as a loading control, numerous other primaries on the same blots. ANTIBODY STORAGE CONDITIONS Aliquoted and stored at -20C upon receipt. SAMPLE PREPARATION RIPA+PI's AMOUNT OF PROTEIN LOADED 20ug ELECTROPHORESIS/GEL CONDITIONS non-reducing, 12% gel TRANSFER AND BLOCKING CONDITIONS 5% milk in TBS-T, 1 hr. SECONDARY ANTIBODY anti-mouse HRP from Amersham, 1hr in 0.5% milk 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? Varied primary antibody concentration between 1:250 and 1:1000 (recommended). Have tried on different cell lines, with and without other antibodies present. ADDITIONAL NOTES GAPDH loading control and all other antibodies tested work great using my current protocol. I have yet to see any band for AurB using this antibody.

Answer

Thank you for contacting us for technical support with ab3609. I'm sorry to hear you are experiencing problems with this antibody. I would expect it to work in HeLa lysate as we have tested the antibody in this lysate and got a nice band. I think the problem could be due to the following: -non reducing conditions. These have not been tried and may not be suitable for this antibody, therefore try reducing conditions - blocking: sometimes the milk prevents the antibody from binding. I would recommend to block in 1% BSA -not enough antibody: try to incubate overnight at 4C in TBST A few more tips to improve signals are to use an Ecl+ or Pierce Super Signal detection kit rather than Ecl;please also make sure that you have plenty of fresh protease inhibitors; check that your secondary works with other primary antibodies. If you still experience problems with reducing conditions, a changed in blocking and an overnight incubation please do not hesitate to contact me and I can offer you a replacement vial or refund as you purchased the antibody in September. Best of luck with your next experiments,

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