I have a problem with blotting on the membrane.. The procedure is as follows: Immediately after termination of electrophoresis, the gel was incubated in the transfer
buffer (0.025 M TRIS, 0.192 M glycine, pH 8.3) with 10 % (v/v) methanol for 10 min. A
“sandwich” consisting of three pieces (the same size as the gel) of Whatman grade 5 paper
soaked in the transfer buffer with 10 % methanol, a PVDF membrane (of the same size as the
gel), that was first soaked in methanol (10 s), then in distilled water (until complete wetting)
and finally in the transfer buffer with 10 % methanol (5 min), the gel and three other pieces of
wet chromatographic paper was assembled. So the first are 5 paper next PVDF membrane and gel and at the end are 5 paper. 10 % methanol contains 20 ml methanol and 180 dH2O and transfer buffer is same. I use trans-blot-turbo and on the displey is no load detected.
Thank you for your advice
Hi there,
I have a stupid question: have you checked the polarity between gel and membrane (membrane has to be between the gel and +...)? And the setting for time and voltage (if too long or too strong, proteins will pass through the membrane)?
I always eliminate SDS from the gel first (by soaking it into water for 2*5min) prior to transfer buffer equilibration.
Hi,
first - not so important for this question, but the PVDF membrane can be soaked a bit longer in the methanol and then you can leave it in the blotting buffer directly, without the step involving the distilled water. Also, 20 % methanol is usually used (but 10 % should work as well).
Regarding your problems, maybe I can help - we had similar problems with Trans-blot Turbo, the problem can be the contacts in the instrument - when you are preparing the stack and then "push" it to close the cassette, some buffer can be leaking and it can then corrode the contacts. So, remove the cassettes from the instrument and clean the contacts inside the instrument as well as on the cassettes.
Hope this helps. Best, Jan.
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