Dear All,

My target protein has two isoforms, one is at 60kda, which give strong WB band, however another is at 45-48kda, which has very weak WB signal, even after 1hr exposure with GE's Amersham ECL+Chemiluminescent CCD camera. Both isoform can be detected by same antibody. I have tried increase primary and secondary antibody dilution but seem doesn't help.

Beside increase amout of loading sample, What method should I use to improve the signal? I am thinking about increasing transfering time, but not sure if it would be too long and over-transfer the target protein through the membrane since the target protein is 45-48kda.

My usual transfer condition is 0.45uM PVDF, wet-transfer, ,towbin buffer with 20% methanol, 100V, 1hr at 4°C.

Thank you