SDS-PAGE problem

[Jayakumar R]

HI...
   I am having a problem with a traditional large format SDS-PAGE that I
have been doing.  I run a 20X23 cm SDS-PAGE gel (7%) at 36mA constant
current for stacking gel and 50mA for running gel for 6-7 hours.  The bands
appear very diffuse (proteins are BSA in all lanes and a high molecular
weight marker).  The electrophoresis temperature is 10C.  The polymerization
temperature is 23C. The concentration of Bis in the gel is 1.6% (double the
normal of 0.8%). The Bis concentration is high since I need to use the gel
for seperating two proteins which differs by only 1 kDa. So the diffuse
bands is going to cause me a lot of problems when I use it for that purpose
which brings me to the second part of this question.  Which conditions are
the best for separating two proteins which differ by hardly 1 kDa?
    Any tips would be gratefully acknowledged since I am at my wits end with
these two strange problems.  Praying.............
Sincerely
jayakumar
 
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Jayakumar R. nair
Dr. John J. McGuire's lab
Grace Cancer Drug Centre
Roswell Park Cancer Institute
Elm and Carlton St.
Buffalo, NY14263.
Tel: (716)-845-8918 (off)
     (716)-833-0704 (Res)
Email: jakku71 at yahoo.com
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