I am having an odd problem with the western wet transfer. Usually, I transfer two gels at the same time, and it works well. But recently, the efficiency of the transfer has decreased. One gel was satisfactory (but not as good as before), and in the other one the marker was transferred, but I observed nothing for the samples. Has anyone had the same problem before? Can you provide any suggestions to avoid this problem?
There are a couple of potential problems that could cause this:
1) Buffers: make sure you make a new stock if you have not already with fresh components (Tris, glycine, methanol, etc).
2) Transfer apparatus: maybe this apparatus has a short or is not correctly distributing charge. Try a new one or another labs.
3) Power supply: same as transfer apparatus.
These would be good starting points. Try to eliminate the variables one by one or you will have a headache trying to figure it all out. Think about anything else that may have changed such as your percentage of gel, gel reagents, protein samples, etc. that may also be culprits.
It's frustrating, but worth it in the end. Good luck,
~Adam
What about the membrane? If you are using PVDF membranes, it can happen that the pre-incubation in methanol was not long enough or the washing afterwards also not.
Hi, in addition to Adam and Steingrimur answer, I suggest to clean your transfer apparatus..use Nitric acid 1N to clean the wires (carefully!!!!!!)and then rinse with ddH20...hope also this helps! good luck!
I would check the buffers. That's usually the first place I look. I switched to a lower quality methanol once and it gave me problems. Does the apparatus get hot during the transfer? Also, you can check the transfer efficiency by Ponceau S staining. It's a removable stain.
Just to add to the headache, I once had an issue quite like that. After checking buffers, transfer apparatus, power supply, and gels, I realized that it was the power source from the wall, of all things. I moved my whole set-up to a different bench on a different circuit, and the problems disappeared. Goes to show you sometimes it's not your fault at all, and that really silly things can make a big difference. Good luck!
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