Measuring protein mobility by photobleaching GFP chimeras in living cells.

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

Research output: Contribution to journalArticle

76 Citations (Scopus)

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)
JournalCurrent protocols in cell biology / editorial board, Juan S. Bonifacino ... [et al.]
VolumeChapter 21
StatePublished - Aug 2003
Externally publishedYes

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Photobleaching
Green Fluorescent Proteins
Fluorescence
Fluorescence Recovery After Photobleaching
Proteins
Light
Confocal Microscopy

ASJC Scopus subject areas

  • Medicine(all)

Cite this

Measuring protein mobility by photobleaching GFP chimeras in living cells. / Snapp, Erik L.; Altan, Nihal; Lippincott-Schwartz, Jennifer.

In: Current protocols in cell biology / editorial board, Juan S. Bonifacino ... [et al.], Vol. Chapter 21, 08.2003.

Research output: Contribution to journalArticle

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