Marty, Interesting question. When we have performed FRET experiments using YFP and CFP, we've noticed that when performing compensation between CFP and the detector used to collect FRET emission from YFP, it can be difficult to correctly compensate both the bright and dull CFP+ cells. That is, if the bright +'s were correctly compensated, the dull+'s were overcompensated; if the dull+'s were correctly compensated the bright+'s were undercompensated. This effect was seen in CFP+ only cells. My thought has been that there is a change in the emission spectrum of CFP dependent upon concentration and/or some kind of spectral quenching effect occurring. Unfortunately, I don't have any firm data to confirm the above hypothesis. Kevin Kevin L. Holmes, Ph.D. Chief, Flow Cytometry Section Research Technologies Branch Bldg. 4, Room B1-38 NIAID, NIH Phone: 301-496-9071 FAX: 301-402-4532 Email: kholmes@niaid.nih.gov -----Original Message----- From: Marty Bigos [mailto:mbigos@gladstone.ucsf.edu] Sent: Monday, August 12, 2002 10:47 AM To: cyto-inbox Subject: gfp spectral shifts? Hi - Does anyone know of shifts in the emission spectra of the various gfp constructs due to intracellular pH, linkage to other proteins, etc.? Thanks. Marty -- Marty Bigos Director, Flow Core Gladstone Institute of Virology and Immunology Building 3 SFGH Rm 509 415-695-3832
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