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Measurement of the Dissociation Time Constant of Plasma Membrane Bound Protein Domains Using GFP Fusion Tags
Published online by Cambridge University Press: 02 July 2020
Extract
We have previously shown that the spatial dynamics of GFP-tagged proteins can be investigated by locally enhancing the fluorescence of GFP using a short UV-laser pulse. 1 We have since developed photobleaching and photoenhancement methods to study binding parameters of GFP-tagged protein domains in intact cells. Here we present an analysis procedure which uses either photobleaching or photoenhancement measurements to determine simultaneously (1) the lateral diffusion coefficient and (2) the dissociation time constant defined for a plasma membrane associated, GFP-tagged protein. The dissociation time constant is defined as the time required for the dissociation of 63% of bound protein and is a functionally relevant parameter related to the binding strength. High affinity interactions are associated with a slow dissociation time constant and low affinity interactions with a fast dissociation time constant. The dissociation time constant of a signaling protein also imposes a critical limit on the cellular response time for signal transduction.
- Type
- Cytochemistry, Histochemistry, Immunocytochemistry, and in Situ Hybridization
- Information
- Microscopy and Microanalysis , Volume 3 , Issue S2: Proceedings: Microscopy & Microanalysis '97, Microscopy Society of America 55th Annual Meeting, Microbeam Analysis Society 31st Annual Meeting, Histochemical Society 48th Annual Meeting, Cleveland, Ohio, August 10-14, 1997 , August 1997 , pp. 167 - 168
- Copyright
- Copyright © Microscopy Society of America 1997
References
1. Yokoe, H. and Meyer, T., Nature Biotechnology 14(1996)1252–1256.CrossRefGoogle Scholar
2. This research was supported by National Institutes of Health grants GM-48113 and GM-51457. TM was supported by a fellowship from the David and Lucile Packard Foundation.Google Scholar