I have a problem in the transferring stage of western blot. We used two casettes in the transferring stage ,and the one near the anode side struggled to transfer the protein. After we changed the location of problematic casette to near the cathode side, and it started to work smoothly again. Addition to the this, constant current was 300mA during the whole process and we were sure the transfer buffer that we use is homogen. If is there anyone struggled with the same problem, I will be very appreciated to learn the solution. But in any case I am open to hear any ideas about this problem. Thank you.
Did the high molecular mass proteins partially transferred didn't transfer at all?
Dilara Oguz ,
Western blot experiments can be very frustrating to troubleshoot since there are so many steps involved and many places where errors can occur. I always suggest to people to include check points within the assay. For example after completing a transfer you should do a S-Ponceau stain to make sure everything transferred. This will save you time and effort to see if the transfer worked or not. But if you are still have issues with the cassette on the cathode or anode side, there might be a short in the wiring within the cassette. I would try with someone elses tank to see if the issues reoccurs.
Hi Dilara Oguz ,
This problem is really a pain in the head :).
This happened to me also! The solution was to buy a new transfer unit. I know it is not a scientific/technical solution, but sometimes is the best, and only, solution.
I recommend dry systems for transfer, like Transblot system Biorad. It improved a lot westerns blots in my lab.
Best,
Jacó
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