Measuring protein mobility by photobleaching GFP chimeras in living cells.

Erik L. Snapp, Nihal Altan, Jennifer Lippincott-Schwartz

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Abstract

This unit describes fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP) using commercially available confocal scanning laser microscopy (CSLM). Photobleaching is the photo-induced change in a fluorphore that abolishes that molecule's fluorescence. The different characteristics of green fluorescent protein (GFP) chimeras in a cell can be studied by FRAP, in which a selected region of the cell is photobleached with intense light. The movement of unbleached molecules into a photobleached region is quantified by imaging with an attenuated light source. The movement of molecules between cellular compartments can be determined by FLIP, in which the same region of a cell expressing a GFP chimera is repeatedly photobleached. The loss of fluorescence from regions outside the photobleached region is monitored to characterize the movement of a protein. Together these two techniques are providing fundamentally new insights into the kinetic properties of proteins in cells.

Original languageEnglish (US)
Pages (from-to)Unit 21.1
JournalCurrent protocols in cell biology / editorial board, Juan S. Bonifacino ... [et al.]
VolumeChapter 21
StatePublished - Aug 2003

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ASJC Scopus subject areas

  • Cell Biology

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