I recently had a custom antibody produced against a trout protein. Based on the amino acid sequence of the protein, the company I used (New England Peptide) selected a region for the epitope and synthesized a 14-mer peptide for immunization of two rabbits.
My protein of interest in other species ranges from 50-80 kDa depending on post-translational modifications. (The expected molecular weight for the trout protein based on aa sequence is about 53 kDa).
My custom antibody appears to detect two major bands, one at about 50 kDa, one at about 75 kDa, in addition to a big smear around 150 kDa. Both bands and the smear do not appear in blots probed with pre-immune serum.
I have no idea which band is my protein of interest and what the smear is all about. Does anyone have any suggestions as to how to determine which band is the "right" one?
Also, my protein is a membrane bound protein - I suspect maybe my protein extraction protocol is inappropriate (I homogenize in a RIPA buffer, spin down at 10,600g for 20 min, then use supernatant for western).
Can anyone suggest a protocol for membrane protein extract that they have used that has worked well (there are so many out there, I don't know which one to try).
Thank you so much!
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