Western Blotting with TransaBind

Anthony Shearing SHEARINA at QUCDN.QueensU.ca
Wed Jun 2 14:13:33 EST 1993


I am trying to use S&S transabind APT membrane to covalently bind
proteins for western blotting and antibody affinity purification,
however, I am not getting any binding.  I ran SDS PAGE gels with 0.25 mg
of purified GST fusion protein accross the gel.  I then washed the gel in
2X200mls 50mM NaPO4 transfer buffer for over an hour and activated the
membrane.  The membrane turned yellow as it ought to upon activation.  I
transfered for an hour on a semi-dry western transfer apparatus at 1.3
mA/cm2 (ie 50mA per 8.5cmX4.5cm gel) at room temp. The membrane turned
red as it should but no protein could be detected by ECL.   I do not want
to play around to much because the membrane is so expensive.  I have
requested info from S&S but any additional advice on using this membrane
for western blotting would be much appreciated.
Anthony Shearing
Shearina at QUCDN.queensu.ca
Queen's University
Ph. (613) 545-6424
Fax (613) 545-6830



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