I have sub cloned some constructs. I added a flag tag before the insert. I have confirmed the constructs by PCR, diagnostic digestion and sequencing. Now, I'm trying to validate them using western blot. I'm expecting a band at 45kDa but I get three bands 45kDa- 50kDa. I tried many antibodies and still the same issue. But, when I do a western for flag tag, I get only one band. Is there a possibility that antibody picks protein with tag and without?
1.There could be possible isoforms which were picked-up by your antibody (not Flag). Your Flag antibody detected only one band, so it is correct.
2. There could be phosphorylation + monoubiquitination (band-1 unphosphorylated; band-2 phosphorylated; band-3 phosphorylated + monoubiquitinated ) however, since your flag band is only one, I would not consider this possibility.
Thanks for your replies. I will attach the two bolts, this first one using anti-flag. I got two bands in the two positive groups and no bands in the other two as expected.
When I used the protein antibody I got two-three bands above and below only in the transduced cells and nothing in cells transduced with empty vector !! I repeated it two times and the results are consistent?
Based on Flag image your flag-protein appears to be equivalent to the top band in your anti-protein antibody blot. In that case the bands between tope green and bottom red "could be" one of the following 1. isoforms, 2. cleaved product, 3. non-specific band.
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