Hi there, Let me answer your questions whithin your letter using ALL CAPITALS. At 02:19 PM 8/12/2002 -0500, you wrote: >Howdy, > >I am having a little problem understanding this whole energy transfer >issue. Say you had a GFP protein and another protein fused with DS >Red. Also say that these two proteins are supposed to interact with each >other. The energy should be transferred from high energy to low >energy. Or GFP to DS Red. >So her comes the questions. Does this strictly result in a loss of GFP >fluorescence and a gain in DS Red fluorescence? BRIEFLY THE ANSWER IS YES. (IN GENERAL, IT IS ALSO POSSIBLE THAT ONLY THE DONOR INTENSITY DECREASES IF THE ACCEPTOR IS NOT A FLUORESCENT DYE, i.e. THE QUANTUM EFFICIENCY OF THE ACCEPTOR IS ZERO. THIS IS CALLED DARK TRANSFER. HOWEVER, IN THIS CASE THE QUANTUM EFFICIENCY OF THE DS Red IS MORE THAN ZERO, SO YOU SHOULD SEE ENHANCEMENT ON THE ACCEPTOR SIDE, IN ADDITION TO THE QUENCHING IN THE DONOR SIDE.) > One of our users went to a >lecture that stated that you actually can get a change in emitted >wavelength(Unpublished data). In the above example the DS Red would now be >Yellow in emission. Is this True? IF YOU MEAN THAT THE EMISSION SPECTRUM OF THE DS Red CHANGES DUE TO THE ENERGY TRANSFER, THEN THIS STATEMENT IS COMPLETELY WRONG. NEITHER THE EMISSION SPECTRUM OF THE DONOR (GFP) NOR THE EMISSION SPECTRUM OF THE ACCEPTOR (DS Red) SHOULD CHANGE DUE TO THE ENERGY TRANSFER PROCESS. THIS COMES FROM THE PHYSICS OF THE PHENOMENON AND THIS IS THE BASIS OF ENERGY TRANSFER CALCULATIONS WHERE SINGLE LABELED SAMPLES (DONOR ONLY OR ACCEPTOR ONLY) ARE USED TO DETERMINE THE SPECTRAL OVERLAPS IN THE ENERGY TRANSFER SAMPLE (DONOR + ACCEPTOR). > Could this be true for confocal but not >for Flow? THE PHENOMENON SHOULD BE INDEPENDENT OF THE INSTRUMENT. THE SENSITIVITY OF THE INSTRUMENTS, HOWEVER, CAN BE QUITE DIFFERENT SINCE DIFFERENT LASERS, OPTICAL SETUPS CAN BE USED IN THESE INSTRUMENTS. IN ADDITION IN THE FLOW SYSTEM THE TRANSFER EFFICIENCY CAN BE CALCULATED ON A CELL BY CELL BASIS, IN THE CONFOCAL MICROSCOPY ON A PIXEL-BY-PIXEL BASIS. > Do we now need to use a different dichroic to find the double >positive cells? PROBABLY NOT. IN THE DOT PLOT, HOWEVER YOU CAN HAVE ONLY THE QUENCHED INTENSITY OF THE DONOR ON ONE AXIS AND THE INTENSITY OF THE ACCEPTOR ON THE OTHER AXIS. IF YOU CALCULATE THE TRANSFER EFFICIENCY YOU CAN CALCULATE THE UNQUENCHED DONOR INTENSITY AND USE THAT ONE INSTEAD. >Am I totally off on this whole issue? Be nice!!! NO, YOU ARE NOT TOTALLY OFF. I DID MY BEST TO BE NICE. > Thanks in advance for the help, marv IF YOU HAVE MORE QUESTION, DO NOT HESITATE TO CONTACT ME. REGARDS JANOS Janos Szollosi University of Debrecen Medical and Health Science Center Faculty of Medicine Department of Biophysics and Cell Biology P.O.Box 39, Nagyerdei krt. 98 H-4012 Debrecen Hungary Phone/Fax: (36) (52) 412-623 E-mail: szollo@jaguar.dote.hu
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