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1) We do not achieve saturation using concentrations of up to 80ug/ml.
2) We compared labelling of bcl-2 transfectants versus neo controls, and
obtained only about 50% increase. Using the same antibody, western blots
showed much greater differences.
We have done a lot of work on this assay, examining different cell types,
batches of antibody, and a wide range of fixation and permeabilization
procedures. Depressingly, the antibody dilution curves stay the same.
My working hypothesis is that the Dako antibody is cross-reacting with
some other intracellular antigens; perhaps another member of the bcl-2
gene family. However, I would be grateful for any ideas from people who
have a reliable method up and running, or who may have any thoughts.
David Hedley
Princess Margaret Hospital/Ontario Cancer Institute
Toronto
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CD-ROM Vol 3 was produced by Monica M. Shively and other staff at the
Purdue University Cytometry Laboratories and distributed free of charge
as an educational service to the cytometry community.
If you have any comments please direct them to
Dr. J. Paul Robinson, Professor & Director,
PUCL, Purdue University, West Lafayette, IN 47907.
Phone: (765)-494-0757;
FAX(765) 494-0517;
Web