Hi,
I am wondering if anyone is familiar with Biorad Trans-blot turbo transfer system. When we purchased this machine, they gave us a 5x buffer, paper stack, and PVDF membrane kit. Indeed, it worked very well.
After we ran out of the kit, we made our own buffer (1x, 48mM Tris, 39mM glycine, 0.1% SDS, ph 9.2, 20% methanol), and used WYPALL X60 Wipers as paper stacks. I noticed the current decreased from 25V 1.3A to 25V 0.8A, and the transfer is not as good as previously since those bands look hollow.
Then we switched to the biorad Filter Paper Backing #1650962. I put 3 layers on each side and the current decreased to 25V 0.4mA. And after 10 mins, the current deceased to 0.2mA. My protein is around 60kd, and no bands at all in the end.
Can anyone spot any issue here?
Thank you
The thickness of the paper stack might be the issue. The driving force for the transfer is the voltage gradient (strength of the electrical field, like in the SDS-PAGE), the current is just a parasitic property of the system (but it's easier to set the current in a defined buffer (defined conductivity) when the transfer system has a defined size, when it's difficult to measure and maintain/reproduce the distance between the electrodes which determines the required voltage. It's basically all connected via Ohm's Law).
The BioRad machine seems to be very similar to other semi-dry blotters like the famous Hoefer TE20 workhorse, so you might try standard semi-dry buffer recipes and calculate the applied current at 0.8mA/cm^2 (filter paper area, not membrane area) and run the setup in constant voltage mode (the voltage would increase, as the buffers get warm and their conductivity decreases, there is a risk to fry your blot and to destroy the apparatus). Run until the pre-stained marker has been transferred, it will take about 2..3 hrs.
Wolfgang Schechinger, thank you. You are right that the thickness of the paper stack is the issue. I tried 1, 2, and 3 layers on each side and increased the SDS to 0.2%. It seems 2 layers on each side gives the best results.
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