Methanol substitute for Western?
Thomas at hastingslab.harvard.edu
Mon Dec 4 18:56:07 EST 1995
"Jennifer L. Potter" <jras at post.its.mcw.edu> wrote:
>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,
>Medical College of WI (no,not Madison!)
Right, binding of proteins is the reason why you usually include Methanol into your transfer buffer. If you have problems with the transfer, 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
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