Hi Sebastian Schmitt

What type of membrane and transfer system are you using?

Some membranes are pre-activated in semi-dry transfer systems (e.g., iBlot), but if this is a fully wet transfer and using PVDF membranes, you'll need to activate the membrane in methanol before transfer (assuming you haven't done this already). However, no activation is needed for nitrocellulose.

I've had problems skipping this step when using PVDF and ended up going through multiple ice packs in the transfer tank, not realizing the membrane was causing resistance in the system.