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I am more than glad help you with some advice in the FRET experiments
but I need more information about your system. (Both instrumental and
experimental.)
You want to measure quenching in a flow cytometer and not bleaching in
a microscope. Is that right?
If you want to measure quenching, you can't determine the transfer
efficiency value on a cell by cell basis, you will obtain only an
average of the energy transfer for the whole population (or gated
population). To perform quenching measurements you have to have very
accurately measured control samples.
E.g. 1. FITC only samples should be measured in the presence of the
same amount of unlabeled antibody as in the case of R.PE antibody! 2.
gating and the calculation of the mean value of the fluorescence
intensity should be the same in case of the doubly and singly labeled
samples. The best thing is to calculate the mean of the fisrt 85 or 90
percentile, in this case the effect of the tail is minimized. Or you
can use the median value. 3. I am not sure, how accurately you can
perform this type of experiments on a log scale, personally I would
prefer the linear scale.
In a dual laser system you have a chance to determine the transfer
efficiency value on a cell by cell basis. So, please write me what
kind of flow cytometer you have!
Sincerely
Janos Janos Szollosi Department of Biophysics Medical University School
of Debrecen Nagyerdei krt. 98 H-4012 Debrecen, P.O.B 39 HUNGARY
Phone/FAX: (36)(52)-412-623 E-mail: szollo@jaguar.dote.hu
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