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I just purchased anti-HDAC3 antibody (ab7030) from abcam. The western blot with NIH3T3 cell lysate showes two band, one is a little bigger than 56KDa and the other is a little smalled than 56KDa. I suppose the samller band is what indicate on the datasheet as 49KDa HDAC3 band. Please see the attached picture.
1.Could you please let meknow why there is a non specific band? any other people showed the same result?
2. I am palnning to use this antibody for ChIP experiment, since this is ChIP grade antibody. Do you think it is okay for ChIP experient since there is non specific band?
Asked on Jun 26 2012
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The mouse HDAC3 has 2 isoforms: 27 and 48 kDa (http://www.uniprot.org/uniprot/O88895).
Also, 2 customers had submitted Abreviews for mouse and WB, one of them for 3T3 cells where they also obtained 2 bands (https://www.abcam.com/index.html?datasheet=7030&tab=abreviews&intabreviewid=22723).
I am not sure how their blot compares to yours since the MW markers are not indicated, but please check the link.
The other review shows one band at 49 kDa for amouse brain sample:
Thus, the bands you see could be specific to thesample you are using.It might be worthwhile including a positive human or mouse positive control (e.g. Hek293 cells). Please keep in mind that the human protein has a different set of 2 isoforms: 48.8 and 49.1 kDa (http://www.uniprot.org/uniprot/O15379).
As for using the antibody in ChIP:
The antibody has been tested in this application and would be guaranteed. If the 2 bands you are seeing are specific to the NIH3T3 cells, then it would be no problem using the antibody in ChIP.
Please see also the Abreviews submitted by other customers: https://www.abcam.com/HDAC3-antibody-ChIP-Grade-ab7030-reviews.html
I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.
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Answered on Jun 26 2012