Lu Falong wrote:
> After transfer, the prestained ladder could be
> seen, but after a brief rinse, most of the prestained ladder was gone
> (Transfer buffer Towbins buffer with 10% methanol and 0.05% SDS).
SDS is used to mobilise large proteins from the gel, methanol to
increase binding of small ones to the membrane. Since they have opposite
effects there is little point in having both present at the same time,
even though a lot of people do that.
Blotting works better with Dunn's buffer (10 mM NaHCO3 and 3 mM Na2CO3,
Anal. Biochem. 157 (1986) 144-153) than with Towbins (and the former is
much cheaper, too).
If you have problems with Ponceau staining try india ink but beware that
that is a permanent stain that interferes with immune detection. Also
some of the fluorescent dyes marketed by Molecular Probes
(www.probes.com) are very sensitive.
In general you can expect better blotting results from PVDF than NC
membranes, as both affinity and capacity are higher. But note that not
all PVDF membranes were created equal, from the limited range I tried I
got best results with Immobilon P (Millipore). YMMV.
