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