Dear Kathy, Let me answer some of your questions within your letter using ALL CAPITAL LETTERS! At 11:46 AM 8/3/99 -0500, you wrote: > >Hi, flowers, > We are beginning to do some experimentation with FRET and I have some >instrumentation questions. First, the experiment: E. coli has been >transfected with both GFP + proteinA and also with BFP+proteinB. Both >proteins are membrane-bound and don't come together unless a stimulus is >applied. > We have been using a 488 (argon) in the primary postion and a 405 >(krypton) in the 2nd position to detect these two fluorophores on a >FACStarPlus. We have been looking at the GFP (530/30) alone off the 1st >laser and the BFP (460/20) off the second. In addition, we have a detector >that looks at any GFP generated by FRET off the second (also a >530/30--with a 505 beam splitter). Because the expression is low we have >been collecting signals with linear amplification--requiring increased gain >settings. THE LINEAR SCALE IS BETTER IF YOU WANT OT CALCULATE FRET EFFICIENCY. FOR EXAMPLE, IF YOU MONITOR THE FRET EFFICIENCY ON THE DONOR SIDE (QUENCHING), IF THE FRET EFFICIENCY IS 10%, THIS WILL CAUSE ONLY 10% DECREASE IN THE INTENSITY OF THE DONOR. IT IS MORE DIFFICULT TO DETECT THIS SMALL CHANGE ON THE LOGARITHMIC THAN ON THE LINEAR SCALE >Question 1: Do we have the right instrument configuration to collect the >respective signals? THE FILTER SETUP SEEMS TO BE O.K. YOU SHOULD KNOW WHAT GFP MUTANTS YOU ARE USING, WHAT SPECTRAL CHARACTERISTICS THEY HAVE. THE CRITICAL FACTORS ARE THE FILTERS, THEY SHOULD BE BLOCKED AT THE EXCITATIONS WAVELENGTH, I. E. 405 NM AND 488 NM. The blue we see (when we can see it, is very dim)., >but the green is significant. (Stupid question --will exciting the GFP >with 488 first, render it unexcitable by the BFP if the beams are spatially >separated?) IN PRINCIPLE NOT, SINCE THE FLUORESCENCE LIFETIME IS MUCH MUCH SHORTER THAN DELAY TIME. IN PRACTICE, THE PHOTOBLEACHING MAY CAUSE SOME PROBLEMS. SINCE YOU WILL RUN ALL YOUR CONTROL SAMPLES UNDER THE SAME CONDITIONS, YOU CAN CORRECT FOR THE PHOTOBLEACHING AS WELL. >Question 2: We are also collecting a ratio (GFP-second beam/BFP-second >beam) but have been having some difficulty because the blue signal has been >so dim. Is this an appropriate measurement? PROBABLY NOT, SINCE ALL THE INTENSITIES SHOULD BE CORRECTED FIRST FOR THE AUTOFLUORESCENCE BEFORE DOING ANY CALCULATIONS OR RATIOING. SAVE THE PRIMARY DATA, LATER ON YOU CAN DO ANY CORRECTIONS, CALCULATIONS. >Question 3: If the transfection efficiency of the BFP-proteinB combination >is bad, can I assume we are SOL? Should the signal intensity of both >fluorophores be somewhat equal to get good measurements? IT IS GOOD TO HAVE SOMEWHAT EQUAL LEVELS OF THE MOLECULES IN QUESTION, BECAUSE THEN YOU CAN PERFROM THE FRET MEASUREMENTS BACK AND FORTH. IF YOU DO NOT HAVE CONTROL ABOUT THE EXPRESSION LEVELS, MOLECULES AT HIGHER EXPRESSION SHOULD BE CHOSEN AS ACCEPTORS. > Any suggestions would be greatly appreciated. > Thanks in advance. >Kathy > > I HOPE THESE SUGGESTIONS WILL HELP YOU IN YOUR EXPERIMENTS. REGARDS JANOS > >-------------------------------------------------------------- >Kathy Schell >Supervisor, UWCCC Flow Cytometry Facility >600 Highland Ave. K4/535 >Madison, WI 53792 >Voice: 608-263-0313 >e-mail: kschell@facstaff.wisc.edu > > > Janos Szollosi Department of Biophysics and Cell Biology University Medical School of Debrecen Nagyerdei krt. 98, P.O.Box 39, H-4012 Debrecen, HUNGARY Fax/Phone: (36) (52) 412-623 e-mail: szollo@jaguar.dote.hu
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