Von Willebrand factor (vWF) is a large glycoprotein, composed of a seried of multumers with MW ranging from about 800 to 20,000 kDa. For analysis of this multimer with western blot, a special electrophoresis and protein transfer are required. In literature (Electrophoresis 2001, 22, 946–949,Clinical application of a rapid method using agarose gel electrophoresis and Western blotting to
evaluate von Willebrand factor protease activity), the transfer protocol was described as: "The protein was transferred overnight (70–100 mA) onto
a polyvinylidine difluoride (PVDF) 0.45 m membrane in a cold room using a Trans-Blot Cell with plate electrodes (Bio-Rad Laboratories, Hercules, CA, USA). The transfer buffer was 0.0025 M/L Tris, 0.0192 M/L glycine, 0.01%
SDS, pH 8.8. "
My question is why the transfer buffer used here is 10 times less than the 1x buffer? How could this 0.1x buffer work for transfer? Thanks!
Hi Shirong
I think this is most likely a mistake by the authors. Although it is customary to use low salt buffers when doing native gel electrophoresis to reduce heat emission during the run, I don't see how this could be applicable to your problem. I assume you are using agarose gels for such large proteinsbut I still don't see how that would cause you to only use 0.1x transfer buffer.
I don't think it will work
Good luck
Jesper
i just realise that these stuff are about 0.8-2.0 (?) MDa. I would strictly follow the original recipe, whatever it means the 0.1x transfer buffer (untill it works, of course).
Hi again
I just realized that the low salt concentration may be a way of increasing the field strength without increasing the current. Transferring such high molecular weight proteins from the gel to the membrane is difficult and would require relatively high voltage. However, increasing the voltage using the standard buffer conditions would probably generate a lot of heat (even in the cold room) which is not good for agarose gels. I guess I would actually try to compare the low buffer conditions with the high buffer.
Best
Jesper
Jesper and Attila: thanks for the helpful discussion. A recipe for transfer buffer I heard was: 10x tris-Glycine-SDS buffer (10ml)+100% methanol (200ml) +DDH2O (790ml). This low salt buffer is out of my understanding regarding the regular transfer procedure! Actually, I had followed it and did not get any band when blotting with anti-vWF antibody. At the same time, when I treated the plasma with 2X Laemmli buffer, 95C, 5min, separated protein by 4-20% SDS-PAGE gel, followed by regular transfer procedure, blotted with same antibody, I got a very nice band at MW of 250kDa, which should be the subunit of vWF. So, my WB procedure sounds good. Since there are many differences between vWF WB by agarose gel from with regular WB, I did not know why I could not get any band from agarose gel protocol although my major concern is the transfer part.
If low salt buffer is reasonable, I may try and compare the low buffer with the high buffer. thanks again!
Shirong
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