Anyone successful with Pharmacia RPAS?
debbritt at brownvm.brown.edu
Wed Jun 12 11:00:56 EST 1996
We have been using PharmaciaÕs recombinant phage antibody system to
construct ScFvÕs from hybridoma cell lines. We succeeded in cloning and
isolating four ScFvÕs, but have had a horrible time producing soluble
antibodies from the clones. There was one promising result using the
ScFvÕs for immunofluorescence, but we have been unable to repeat it. We
have had no luck at all doing Western blots, either running the ScFv prep
on the gel, or using it as the primary antibody with the Pharmacia anti-E
tag monoclonal as the secondary antibody. Has anyone managed to produce
soluble antibodies using this system? I would be happy to hear from you,
just to know it can be done. At this point, we cannot tell if the problem
is in ScFv expression, purification or detection. A good positive control
would be a great help. Would anyone who has the system working be willing
to share their ScFv plasmid? (This would be used strictly as a positive
control to test our procedures). Any feedback would be greatly
appreciated, as we are extremely frustrated!! Thanks in advance.
Deborah Britt, Ph.D.
RI Hospital/Brown University
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