Hello,

1) Check the gels- if you make them yourself, maybe there is an error in this step- is the % right for your proteins of interest, are you letting them set long enough between steps and in optimal temperature; Also check the TEMED and acrylamide best before dates and if they are kept in /-+ 4-8 degrees;

2) Check, if you use optimal membrane type- PVDF are better in most cases but if proteins of interest have sensitive bonds- cellulose might work better;

3) If you make solutions yourself, concentrations and pH also play a big role- check how long ago you prepared solutions and if they are kept in optimal conditions (out of sunlight, cold-ish and dry place) and haven't, for example, crystalized or changed color;

4) For transfer step- check if you add enough transfer solution (usually before closing compartment i 'slush' a bit of solution by pressing gently on the compartment lid so that it gets evenly to all membrane sandwich);

Wet the filter papers before laying on membrane and gel, make sure there's no air bubbles;

Also- chek out the transfer step settings- maybe the current is too weak or too strong for proteins to go trough.

I have the settings on: LIST->BIORAD->2 mini gel (i usually do 2 gels in one compartment)-> Standart (25V-1.0A-30M)-> RUN-> A run, for some 30 minutes

If proteins with higher weight even in protein ladder did not transfer, then most likely they dont have enough time to go trough, so if possible, play around with settings, try setting longer time or higher current

Larger proteins above 70 kDa transfer better with a wet system. Use fresh buffer and increase transfer time. Proteins above 300 kDa require at least 4 hours in the refrigerator

Thanku Af D. for your response

Thank you, Beatrise Luīze Revina, for adding your response

I have the same problem, have you found a solution?

I use all BioRad reagents, including Trans-Blot Turbo 5x transfer buffer. I have tried changing the transfer settings, such as more time, but I see nothing on my PVDF membranes and I see protein in the gel after transfer, especially the high molecular weight, and the small molecular weight does not seem to be anywhere, neither in the gel nor on the membrane.