 |
 |
 |
 |
I want to thank all of the people who have sent me advice on
detecting IL-8 receptors. I have received a number of personal E-mail
messages, as well as messages that were also posted directly to this list.
I would like to suggest that all messages get posted directly to the list so
that everyone can participate.
A number of people commented on the use of the R&D Flurokine kits.
We have unfortunately tried several of these Flurokine kits in the
past. They are extremely expensive, and put simply, they just don't work in
most cases. The PE-conjugated cytokines show very poor fluorescence, and
they are often non-specifically sticky on PCR-receptor-negative cell lines.
The biotin-conjugated cytokines (followed by SA-PE) can show better shifts,
but our SA-PE-alone controls were often extremely sticky on PCR-negative
cell lines. We have also seen significant lot-to-lot variability. At @
$500 a shot (good for maybe 5 failed experimtents), it just is not worth it.
I have seen very few publications (aside from R&D data) showing "good"
results with these reagents.
We have also tried the very expensive experiment of competing a
flurokine (G-CSF-PE) with a preincubation of 100-fold excess of recombinant
G-CSF (either E.coli or yeast produced). Only a very marginal inhibition of
flurokine binding was seen. A new kit (shipped on ice) provided by R&D
provided somewhat better results. I realize that our cells may express very
few receptors, and thus show weak shifts, but there has to be a reasonable
way to prove that these kits work specifically, and there needs to be some
resonable quality control. This is clearly a technique of last resort.
We have also had numerous conversations with Tech-support at R&D.
All they say is that it works fine for them. One problem is that they ship
these kits at ambient temperature, even on the hottest summer days. On cold
days, they may encounter several freeze-thaw cycles.
Have a bright and cheery day! Oh, and Happy New Year!!!
--Rich
Richard Moldwin, M.D., Ph.D.
Assistant Professor of Pediatric Hematology/Oncology
University of Chicago Hospitals
5841 S. Maryland Ave., M/C 4060
Chicago, IL 60637
E-mail: rmoldwin@midway.uchicago.edu
Phone 312-702-6808
Fax 312-702-9881
|
|
|
|