c-myc-tag/9E10 detection problems
kjaer at biobase.dk
Sat Nov 18 04:15:14 EST 1995
Hi netters !
We use the Nissim ScFv-phage displayed library from Greg Winters lab. A number
of clones have been found toward different antigens. They react positively
in phage-ELISA and phage-western blots, but when the E.coli strain is switched
from the suppressor strain TG-1 to the non-suppressor strain HB2151 to allow
for soluble scFv production, the antibody fragments doesn't work neither in
ELISA nor in western blots.
However, when a crude lysate of the scFv producing bacteria is run onto a
SDS-gel, blotted and probed with 9E10 antibody, that specifically recognize
the c-myc tag present at the c-terminus of the scFv, there is a nice speci-
fic band present at the right size. This means that the expression of the
scFv is okay.
So why doesn't the ELISA and Western blots work, when scFv's are used as
primary antibody ?
We tend to believe that the 9E10 antibody mostly recognizes denatured c-myc
tags and as the scFv, when used as primary antibody, is not denatured the
c-myc-tag is somewhat difficult for the 9E10-antibody to find.
Still, other people seem to have it working, so why don't we????
Any thoughts and suggestions are highly wellcome
Thanks in advance
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