I tried a transfer on PVDF with Tris-Glycine buffer containing 10% methanol and 0.1%SDS at 25V for 16 hrs. My protein ladder (HiMark from Invitrogen) went through so I was not sure of the molecular weight I blotted for, i could see a weak band at the top of the membrane but it is very subjective.
Hi Dinesh, thanks for the suggestion of reducing the methanol content, I will try that. I used the 3-8%Tris Actetate gels.
We normally use 20% methanol for the transfer without SDS, 200KDa bands are transferred in 1h 30min at 300mA in refrigerated conditions from 10% gels (wet transfer). Alternatively we transfer O/N at 30V. Given that your gel is way less concentrated I don't see why it should not work in the same conditions or slightly different, for example 50V 15h. If the 460KDa band of your gel is on the membrane you protein should be there too: in this case I would double check the Antibody and the TBST buffer you are using.
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