To all: We have found that Alexa647 seems to match the HeNe laser the best, with the best compromise of absorption and emission spectra, at least for the FACScalibur filter set and probably for most sorters that are set up for APC. It certainly gives much better signal than 633, conjugating the same protein preps. While 633 gets excited more optimally by the HeNe, its Stokes shift is small and much of its emission is cut off by the typical filters used for APC/FL4. Although 647 has almost the same spectral qualities as Cy5, for some reason we seem to get superior reagents from the Alexa dye. I would also point out that (finally) Molecular Probes is now selling the bulk hydroxysuccinimide ester reagent, making it much more economical for those who want to do a number of conjugations. However, none of the Alexa dyes is going to be as bright as APC, so if brightness is paramount, you will still want APC. A question to Pete: are you distinguishing the 630 and 660 signals from the same excitation simultaneously? If so, what filter sets do you use and what are the compensations, etc. Mark Shlomchik, MD, PhD Associate Professor of Laboratory Medicine and Immunobiology Yale University School of Medicine At 4:41 PM -0500 2/1/02, Howard T Petrie wrote: >Having spent many years conjugating many antibodies to >phycobiliproteins, I can understand why you wouldn't want to do it >yourself (although the results are ALWAYS better). For the last >couple of years, however, we've been using Alexa633 instead, and I >have to say that even using the straight-out-of-the-box protocol >supplied by Molecular Probes, the results are quite good (if you have >enough antibody to spare and want to test a couple of chrome:Ab >ratios the results can be even better). I highly recommend giving it >a try, it takes about one hour of hands-on time and about three hours >in total. > >I should mention that Alexa660 works beautifully as well for flow, in >fact better than 633 (I'd love to hear somebody at either MP or one >of the equipment manufacturers explain why this should be the case; >neither the optics nor the spectra would predict it). In fact after >years as a dye laser loyalist, we now use HeNe exclusively, since we >can simultaneously excite and detect A633 and A660 instead of APC and >TR. Given the utter simplicity of conjugation of the Alexa dyes, >it's worth considering. > >> To All: >> >> I am in need of getting two monoclonal antibodies conjugated to APC. >> If you have had this done by some company/lab and liked the result >>would you please let me know. >> >> thanks >> >> Jim Houston > >-- > > >Howard T. Petrie, Ph.D. >Head, Laboratory of Developmental Immunology >Memorial Sloan-Kettering Cancer Center >Box 341, 1275 York Avenue >New York, NY 10021 >phone (212)639-2149 >fax (212)794-4019 -- Mark Shlomchik, MD, PhD Associate Professor of Laboratory Medicine and Immunobiology Yale University School of Medicine 203-688-2089 203-688-2748 (fax) mark.shlomchik@yale.edu
This archive was generated by hypermail 2b29 : Sun Jan 05 2003 - 19:25:59 EST