TY - JOUR
T1 - Measuring protein mobility by photobleaching GFP chimeras in living cells.
AU - Snapp, Erik L.
AU - Altan, Nihal
AU - Lippincott-Schwartz, Jennifer
PY - 2003/8
Y1 - 2003/8
N2 - 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.
AB - 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.
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M3 - Article
C2 - 18228432
AN - SCOPUS:39049110236
SN - 1934-2500
VL - Chapter 21
SP - Unit 21.1
JO - Current protocols in cell biology / editorial board, Juan S. Bonifacino ... [et al.]
JF - Current protocols in cell biology / editorial board, Juan S. Bonifacino ... [et al.]
ER -