Shaun D. Black
shaun at uthct.edu
Fri Mar 20 16:29:46 EST 1998
Stephane (stephane.ory at ens-lyon.fr) wrote...
>we have difficulties to transfer high molecular weight protein. Does
>anybody have an effective transfer method for high molecular weight
We and a few labs discovered the solution to this a couple of years ago.
In fact, this is one of the many useful tricks I have detailed in the
SDS-PAGE and Western Blotting book chapter we recently posted about on
Method and Reagents. For Western Blotting of all proteins (including huge
molecules up to about 1,000,000 molecular weight), we use standard Towbin
buffer (tris, glycine, 20% methanol) with 0.01% SDS (w/v). This may seem
like a trivial change, but 0.01% SDS works wonders with most all proteins.
More SDS is bad, and less SDS is bad. After fooling around with this a
bunch, we've settled on this recipe and find that it basically works for
everything. I hope this helps.
P.S., the reference for our recent book chapter where this and much more
is detailed, is:
"Diagnostic Application of a Multiple-Channel Immunoblot Apparatus"
S.D. Black, in Methods in Molecular Medicine Vol. 13, Molecular
Diagnosis of Infectious Diseases (U. Reischl, ed.) Humana Press,
Totowa, NJ (USA), pp. 373-396, Chapter 22 (Nov. 1997)
= Shaun D. Black, PhD | Internet e-mail address: shaun at uthct.edu =
= Dept. of Biochemistry | University of Texas Health Center at Tyler =
= Tyler, TX 75710 | World Wide Web: http://www.uthct.edu =
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