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Direct FACStar titration of the TRITC conjugate gave a half-maximal
binding at a concentration >= 1/25, less than 10% of the input binding
activity. However, today we found that _indirect_ FACScan titration
gives 1/250, exactly the expected activity. Thus, NO ANTIBODY WAS
INACTIVATED. We are at a loss to explain the direct titration result
since the A550/A280 was 0.22. We will repeat the titration, and also
try using Molecular Probes carboxytetramethylrhodamine succinimidyl
ester for our next try.
It turns out that the second antibody mentioned was entirely inactivated
during the purification process (before conjugation), presumably by
low pH elution. Thus, TRITC was unrelated to its problem.
--
/* - - - - - - - - - - - - - - - - - From - - - - - - - - - - - - - - - - - -
Eric Martz emartz@titan.ucc.umass.edu Professor of Immunology
Voice 413-545-2325, FAX 413-545-1578
Morrill IVN Rm 203, Dept Microbiol, Univ Mass, Amherst MA 01003 USA
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - */
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