From: Jim Grobholz (jgrobholz@omegafilters.com)
Date: Thu Jul 30 1998 - 08:49:20 EST
rsGFP (ex 490/em 509) can be analyzed in combination with the "yellow" mutant, YFP (ex 513/em 527) using a single 488nm line - see Lybarger et al Cytometry 31:147-152(1998). A 525 shortpass dichroic beamsplitter (SP) in the emission path separates the GFP/YFP signals. The GFP signal is transmitted to one PMT(FL1) which has a 510/20 emission filter, while the YFP signal is reflected to another PMT(FL2) which has a 550/30 emission filter. Clontech sells vectors for both spectral variants of GFP (EGFP and EYFP), and Omega Optical sells a stock filter set which is comprised of the components listed above or with a longpass dichroic (XF500). Jim Grobholz Lucy Brown wrote: > > We have a couple of users who would like to use two reporter genes in > one construct. One has tried GFP(red shifted) along with BFP both > were purchased from Clonetech. We have a three laser bench--a HeNe, > and Innova 90 set at 488, and an Innova300 which we can convert from > UV to 514. We were able to see GFP exicitation but saw nothing from > BFP excited with multiline UV 200mW of power and a 450DF65 as a > collection filter. I have been told that BFP is easily photobleached > and just not a good variant to use. > > My question is: Has anyone used in combination with GFP a second > variant and if so what was the excitation wavelength, the laser power > and collection filter used? > > Thank you for your help > > Lucy Brown > Beckman Research Institute/City of Hope Med Center > Duarte, Ca > 626-359-8111 X3306 > lbrown.coh.org -- @@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@ Jim Grobholz Phone: (802) 254-2690 Product Manager - Microscopy Fax: (802) 254-3937 Omega Optical, Incorporated 3 Grove St., P.O. Box 573 Brattleboro, VT 05302-0573 Email: jgrobholz@omegafilters.com http://www.omegafilters.com @@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@
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