Hi all, I am trying to do the western blot for detecting high molecular weight protein ATR (300kDA) together with other proteins with molecular weight of 100kDA, 56kDA, and 35kDA at the same run.

My lab currently used the Biorad Mini Trans-Blot® Electrophoretic Transfer Cell for transferring the protein from the gel to the membrane (80V, 0.35A, 1 hour, in ice). I tried using this method, the lower kDA protein seemed detected fine but it seemed that the ATR (300kDA) was not transferred properly. I am currently using Bio-Rad’s 4–20% Mini-PROTEAN® TGX Stainfree Precast gel and 1x transfer buffer (25 mM Tris, 192 mM Glycine, 20% methanol, 0.05% SDS).

Does anyone has any experience in handling this similar situation (detecting high molecular weight protein)? I would appreciate if anyone can share your experience. Thank you!