Western blues

clarosa clarosa at biocomp.unl.edu
Fri Jul 6 11:49:57 EST 2001


Try looking at the PI of the protein.  The buffers mentioned work better on
acidic proteins or only with proteins made negative by addition of sds
If you are doing semi-dry blotting you can cut out mylar plastic masks (thin
clear plastic) to cover the areas outside of the gels.   I do this routinely.
Some semi dry transfer apparatii will tolerate 2x the amperage per square inch,
I use the hoeffer gadget which has metal gridded plates that seem to transfer
the heat well.  I work with a 130 kDA protein which transfers poorly but I have
found that I can push the protein out by using the higher amperage increasing
the transfer time beyond 2 hours and using PVDF as opposed to nitrocellulose
will help your yield.

Sounds like your using Tris HCL sometimes, rather than Tris Base because Tris
Base never gives acidic pH but Tris HCl will.  There are alternative buffers
which may
might work better, CAPS.

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:
>




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