I have performed Western Blotting many times for getting the five bands for opa1 isoforms in HeLa and HeLa rho zero cells by running the samples on 12,15 and 18% gel for 8,10,12,14,16 and 18 hrs. However, this time I got just three bands instead of the five bands that I usually get. I am using PVDF membrane with methanol in trans buffer and 400mA current. What should I do next to get my respective five bands?
I'm certain there are many possible "technical" reasons this could happen, such as the relative size of the bands, and the antibody's ability to detect each isoform. I have a more general question, however. Are you certain each isoform is expressed under your growth conditions? I do not know the Opa1 gene, but the gene isoforms that I have worked with in the past are not constitutively expressed. Can you, for example, amplify the mRNA from each isoform under your growth conditions?
Thanks Mark for your valuable answer. I will also check out the protein expression at mRNA level to confirm their expression.
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