We transferred proteins from SDS-PAGE onto nitrocellulose using standard homemade Towbin/transfer buffer (25mM Tris, 192mM, 20% methanol) and whatmann filter papers. The problem is that compared to the BioRad transfer packs, the amperage stays so low at 0.1A during transfer instead of 1.1-1.3A (protocol used is High MW single mini gel) when using our homemade consumables. We even tried adding SDS (0.1%) to the buffer but still the amperage reads 0.1-0.2A.
Does anyone have the same problem when using homemade consumables for the transblot? If not, do you mind sharing the type of buffer you used?
Use the following Electroblotting buffer
Dissolve 7.5 g Tris base and 36 g Glycine in 2.25 litres water and check that everything is dissolved, it usually takes an hour (good for lunch break), then add 250 ml Methanol and mix well. I can't tell why your transfer buffer gave problems, and I don't think it is the fact that you use 20% methanol whilst we use 10%. Double check what you actually did when you made the solution (I means stat the amounts of grams per litre, not the theoretical molarities).
@ Vivian: I had the problem with 3M Whatman and oder filter paper. This seems due to a high resistance. With more fabric like filter paper as mentioned by Aman in the other thread (Wypall X60), I get now 0.5 A and 25V in the beginning of my 30 min transfer. After 10 min the ampere increases to 1 A and the Voltage goes down to 17-18 V.
However, for semi dry blotting I prefer Kyhse Anderson 3 buffer system with slightly modified conditions. I use only 5% MeOH in the Cathode buffer and 0.1% SDS.
Thank you Jurgen and Jens!
I've used some local brand kitchen fiber pads (wypall isn't sold in my country) and increased SDS content to 0.5%...and I was able to achieve 1.3A. However, the blots were not successful with certain antibodies, most likely due to sds. I will give the buffer compositions you mentioned a try and thanks again for your answers.
Vivian
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