Methanol substitute for Western?
Frank Chen
I got a question for you.
Currently, I am using methanol in the blotting buffer for
transfering protein onto nitrocellurose membrane. It generates a lot of
"not for sink" aqueous waste. I am wondering if I can use ethanol as the
substitue. Please let me know if you have tried it.
Not long ago, someone posted the substitute of ethanol for
mathanol for fixing gels after electrophoresis. It reduces the methanol
waste a lot for us (and the environment). But, that was only for fixing the
gel. I wonder it may be different in this case.
Your oppinions will be appreciated.
Sincerely,
Frank Chen
Digs
running buffer if you use a semi-dry transfer apparatus.
Paul Digard
Virology
Cambridge
Jennifer L. Potter
Our lab does quantitative Western blotting and we were having problems
getting complete transfer of our proteins.
We simply eliminated the MeOH from our buffer and have had great
transfer ever since (43.2 g Glycine, 8.92 g Tris Base, 4 L DW).
In Western blotting, many competing things are happening (i.e. SDS on the
proteins is what makes them move onto the nitrocellulose but SDS also
decreases the binding affinity of the nitrocellulose)
It's probably best to work out conditions empirically.
So I guess I don't know the answer to your question if EtOH is an okay
substitute for MeOH, but you may not need to use it at all.
Hope this helps,
Jennifer Potter
Grad Student
Medical College of WI (no,not Madison!)
Thomas Urbig
Right, binding of proteins is the reason why you usually include Methanol into your transfer buffer. If you have problems with the t=
ransfer, you can increase your transfer time or include SDS in a small concentration or, as you did, omit the MeOH from the buffer. =
Ethanol will make the transfer worse (it will probably increase the binding, but make the transfer even worse). If you really don't =
want to use MeOH and you have problems with the binding ability of NC use one of the NC-membranes with higher binding capacity.
Hope that helps
Thomas