hi Diana，

I think you should transfer the protein on the Gel completely to the PVDF membrane in order to minimize the unspecific bands. I used the same antibody as you and shared the similar experience as you. So, you can try again

Best

zhiyuan

Hey Zhiyuan. Thanks for replying so quickly. So what do you mean by transfer the protein on the gel completely? All my proteins transfer to the membrane normally, as I do the Ponceau staining after and also I can see my marker.

bw

Diana

Dear Diana,

I mean that you should transfer the band above 170KD more completely. As to Ponceau staining, maybe it is not necessary if the marker has transferred to the membrane.Some high MW protein need longer tranferring time or you could prepare the 8-10% seperation SDS-PAGE for better seperation high MW protein.

best

zhiyuan

We use same antibody but we get one band. You need to have longer time for transfer (about 2.5 hrs, 200 to 250 mA)  and lots of cool packs around to not let temperature increase. We use marker that has a band at 250 kDa to confirm that transfer is complete.