Dear all,
I am doing WBs for the evaluation of Neurogenin-3 protein levels in mice brain lysates using a polyclonal antibody from Merck and I have seen that it shows multiple bands instead of one single band at about 32 kDa (so it is reported in the datasheet). In particular, I can see a very intense band at about 40-50 kDa and a smaller one near 110 kDa. The band I am supposed to see, anyway, is at 32 kDa and it looks very faint.
Browsing on other antibodies, I have seen that this protein can be very varied, going from 23 kDa to 50 kDa, and usually the companies show images that contain multiple bands, although highlighting the desired band.
So, do you have any experience with this protein, in particular with brain lysates? Is it possible that my antibody is non-specific and that it has recognized actine as well? I attached you a photo of one of the WBs, unfortunately without marker (gel is at 12%).
Thank you in advance for your help!
Alessandro
Alessandro,
as your antibody is polyclonal, the preparation may recognize several epitopes, which not necessarily are unique, so other proteins may show on your blot. This even may happen with peptide antibodies or monoclonals.
Posttranslational modifications and alternative processing/splicing also may add diversity to your blot.
If possible, include a positive control (e.g. recombinant protein) or characterize the bands by mass spectrometry (ask proteomics people for assistance)
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