I am working on exosome characterization by identifying the CD markers through Western blotting. Recently in one of the experiment I have come across a strange problem in the transferred PVDF blot. After the immediate transfer of the bands from gel to membrane, I have seen a definite band at 50kda. But after probing with Antibody of my interest, the that band which is at 50kda length is also my protein of interest, has disappeared during chemidoc imaging.
Could anyone plz help me with this?
Do you mean, the transfer was ok and it was confirmed by Ponceau staining or stain-free monitoring? but after antibody incubation and probing sequence, the band was not visible...? If these statements are correct Maybe the protein is not the target one you are interested in...
Dear Mr.Ismail
Yeah that might have happened and i might have assumed that my protein of interest is present for sure.
It may be that what you saw was not your protein of interest, or that your protein does not stick to the membrane well and comes off with the detection procedure. You could try a dot blot of your protein to test the latter.
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