Why no SDS in your transfer buffer? At one time, I got enhanced transfer when I included SDS in the transfer buffer; I assumed that I was probably stripping the SDS off the protein during the electrotransfer, and the SDS in the buffer kept the protein in the negatively-charged SDS-complex.
Also, what is the pH and concentration of your Towbin buffer?
Vernon A. Shoup
Regeneron Pharmaceuticals, QC
Rensselaer, NY 12144
vernon.shoup@regpha.com
(518)488-6012
--------------------------------------
Date: 4/14/98 11:46 AM
To: VERNON SHOUP
From: Jacek_Mozdzanowski-1
To: abrf@aecom.yu.edu
cc:
From: Jacek Mozdzanowski-1 @ SB_PHARM_RD
Date: 14-Apr-98 01:02:36 PM
Subject: Eblot
Categories:
Larry,
Two factors will influence transfer yield the most: acrylamide percentage
in the gel and SDS concentration in the gel and electrophoresis (not
transfer) buffer. Try casting 6% gel using 0.2% SDS concentration in the
gel and then run electrophoresis using electrophoresis buffer containing
0.2% SDS. Do not presoak the gel prior to transfer and try 0.5x Towbin
buffer (Tris/glycine) containing maximum 10% methanol. Do not add SDS to
transfer buffer.
Some proteins will not transfer with 100% yield regardless the conditions,
but the above steps should improve the yield.
Jacek Mozdzanowski
Bioanalytical Sciences
SmithKline Beecham