Ds-red has a secondary excitation shoulder around 488 nm with an extinction coefficient about a third of that at 558 nm. The problem is that Ds-red is not a good chromophore (yet). The extinction coefficient at 558 nm is around 27.000 +/- 3000 and the quantum yield appears higher than published, but not higher than 35-40%. Saverio Alberti Head, Lab. of Experimental Oncology Department of Cell Biology and Oncology Consorzio Mario Negri Sud 66030 Santa Maria Imbaro (Chieti), Italy Phone: (39-0872) 570.293 FAX: (39-0872) 570.412 E-mail: alberti@cmns.mnegri.it On Tue, 9 May 2000, Mark A. KuKuruga wrote: > > Joseph, > The ECFP form will not work with your typical flow setup. You'll > require a Krypton laser, with optics to provide a 407nm excitation > line. Look at J. Dezz Ropp et.al. (Cytometry 21:309-317, 1995; Aequorea > green fluorescent protein analysis by flow cytometry) as a good early > reference describing these parameters. > Keep in mind, though, that this difficulty may be overcome by using the > recently introduced DsRed variant (Clontech). While the peak excitation > is shown at 558nm, the claim is that it is also nicely excitable at > 488nm. > MAK. > > Joseph Webster wrote: > > > I have a user who wishes to run Clontech EGFP and ECFP together; we > > can run EGFP O/K but we have not yet tried ECFP & I'm dubious.... > > > > Has anyone used ECFP (cyan) in a flow cytometer? > > If so, what laser & wavelength excitation? > > > > The Clontech website says excitation is 433 with a secondary > > absorption peak at 453. > > No spectra shown, so I don't know how broad the absorption curve > > might be. > > We don't have any 433 line available, would the argon 457 line work? > > > > Any ideas &/or experience? > > > > Thanks, Joseph. > > -- > > Joseph Webster, Flow Cytometry Facility > > Centenary Institute, Sydney AUSTRALIA. > > -- > Mark A. KuKuruga, Managing Director > University of Michigan Core Flow Cytometry > <http://www.cancer.med.umich.edu/flow_cytometry> > phone: 734-647-3216 fax: 734-936-7376 > kukuru@umich.edu > > >
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