Doing phospho-STAT5 Western on NK92 human cell line. I keep getting two bands, one fainter than the other. Online searches imply that this might be STAT5a and STAT5b, but the difference in amino acids between these two is 12, and the bands look a little too spread out to just be a difference of 12. Does anyone have any insight? The band in the middle of the gel is off target binding.
Have you tried probing for the normal STAT5a to see if the bottom band is the unphosphorylated version? Phospho-antibodies, depending on how they're purified, can have some non-phospho antibodies in the vial/batch - especially if they're purified by protein A/G only and not reverse peptide purified. It might be worth checking that out in the datasheet, but also BLAST the sequence of the peptide that was used to generate the phospho-antibody.
12 amino acids at around 110 Da per amino acids is around 1.5 kDa so it may be possible to be the other isoform.
Thanks Albert Lee
I also do total STAT5 phosphorylation and I get a double band as well. 1.5kDa difference could be what I am seeing.
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