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?