klenchin at REMOVE_TO_REPLY.facstaff.wisc.edu
Fri Jun 22 19:29:37 EST 2001
tdschr0 at pop.uky.edu ("Todd Schraw") wrote:
:Having similar problems with transfer efficiency of a 200 kDa protien.
:Referencing "Molecular Cloning, A laboratory Manuel " (Sambrook, Fritsch,
:Maniatis) They suggest an effective range of separation of
:SDS-Polyacrylamide Gels as follows:
:15% = 12-43
:10% = 16-68
:7.5% = 36-94
:5% = 57-212
These numbers are significantly off IMHO. For example, 7.5% is
too low for 36K and there is absolutely no need to deal with
miserable 5% when you need a good separation in the range
:They also recommend the following Transfer Buffer:
:39 mM Glycine
:48 mM Tris Base
:0.037% SDS (electrophoresis grade)
:They have the transfer running with a current of 0.65 mA/sq. cm. of gel
That would be per sq.cm of gel's cross-section (width x thickness),
not just "gel".
:Another aspect that they repeatidly point to is the possiblity of a short by
:using nitrocelluse or stack paper that is larger than the gel itself. The
:make a big deal about having these exaclty the same size as the gel.
This is simply so wrong, it's not even funny. I never woudl have
thought to find this in Maniatis.
For 220K protein I would try any combination of the following
(in the order of preference; if first does not help, combine it
with the next): 6% gel, strongly alkaline buffer (like CAPS pH
~10), twice longer than normal transfer time (make sure cooling
is OK), 0.01% SDS in transfer buffer.
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