Guide for doing FRAP with Leica TCS SP2 - Biocenter

FRAPFinal RemarksWe would like to recommend beginners, who want topractice FRAP experiments, to use a system similar tothe FITC-dextrans as demonstrated here before tryinglive cells. Keep in mind that in particular the bleacharea size, the laser intensities, FRAP modes, bleachformat, scan speeds as well as the iteration numbersand intervals for bleach and post-bleach acquisitionhave to be optimized for each cell type and protein ofinterest. The results given here were obtained byevaluating just one data set. For meaningful resultsthe experiment should be repeated at least 10-20 timesto get an estimate for the standard deviation. Repeatingthe FRAP experiment using the same cell andbleach ROI can also reveal potential phototoxicity, ifthe results are not reproducible. In such a case theFRAP protocol has to be further optimized (s.a.decreasing the laser power).References1. Phair, R.D., Misteli, T. (2000) High mobility of proteins in the mammaliancell nucleus. Nature. 404:6042. Beaudouin, J., Gerlich, D., Daigle, N., Eils R., Ellenberg, J. (2002)Nuclear Envelope Breakdown Proceeds by Microtubule-InducedTearing of the Lamina Cell 108:83-963. Axelrod, D., Koppel, D.E., Schlessinger, J., Elson, E., Webb, W.W.(1976) Mobility measurement by analysis of fluorescence photobleachingrecovery kinetics. Biophys. J. 16:1055-10694. Phair, R.D., Misteli, T. (2001) Kinetic modelling approaches to invivo imaging. Nat Rev Mol Cell Biol. 2:898-9075. Snapp, E.L., Altan, N., Lippincott-Schwartz, J. (2003) Measuringprotein mobility by phtobleaching GFP chimeras in living cells.Curr. Prot. Cell. Biol. 21.1-21.1.246. Ellenberg., J., Siggia, E.D., Moreira, J.E., Smith, C.F., Presley, J.F.,Worman, H.J., Lippincott-Schwartz, J. (1997) Nuclear membranedynamics and reassembly in living cells: Targetting of an innernuclear membrane protein in interphase and mitosis. J. Cell. Biol.138:1193-12067. Siggia, E.D., Lippincott-Schwartz, J., Bekiranov, S. (2000) Diffusionin inhomogenous media: Theory and simulations applied to a wholecell photobleach recovery. Biophys. J. 79:1761-17708. Lippincott-Schwartz, J., Altan-Bonnet, N., Patterson, G.H. (2003)Photobleaching and photoactivation:following protein dynamicsin living cells. Nature Cell Biology Suppl:S7-S149. Braeckmans, K., Peeters, L., Sanders, N.N., De Smedt, S.C.,Demeester, J. (2003) Three-Dimensional Fluorescence Recoveryafter Photobleaching with the Confocal Scanning Laser Microscope.Biophysical Journal 85:2240–225210.Misteli, T., Gunjan, A., Hock, R., Bustink, M., Brown, D.T. (2000)Dynamic binding of histone H1 to chromatin in living cells. Nature408:877-880Copyright © Leica Microsystems Heidelberg GmbH • Am Friedensplatz 3 • D-68165 Mannheim, Germany • Tel. +49 (0) 6 21-70 28-0 • E-Mail: clsm.support@leica-microsystems.com LEICA and the Leica Logo are registered trademarks of Leica IR GmbH.Order no. of the edition: 1593104005 • August 2004@ www.confocal-microscopy.com