Western blues

Todd Schraw tdschr0 at pop.uky.edu
Fri Jun 22 16:06:37 EST 2001


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

They also recommend the following Transfer Buffer:
39 mM Glycine
48 mM Tris Base
0.037% SDS (electrophoresis grade)
20% Methanol

They have the transfer running with a current of 0.65 mA/sq. cm. of gel for
a period of 1.5 to 2 hours.

In several experiments I have seen better transfer of higher molecular
weight proteins with the addition of SDS. Also we have had problems with
poor grades of Tris base that give acidic pH once dissolved in water instead
of the usual ~8.5.
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.

Hope this is informative. Let me know if it works for you.

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