Box 8.1Fluorescence recovery after photobleaching (FRAP)

Visualization of protein mobility in living nuclei.

FRAP is perhaps the most informative of the various innovative microscopy techniques that have opened up our understanding of nuclear substructure. It has enabled, for the first time, the movement of proteins to be visualized inside living nuclei, the resulting data allowing biophysical models of protein dynamism to be tested.

The starting point for a FRAP experiment is a nucleus in which every copy of the protein of interest carries a fluorescent tag. Labeling the protein molecules in vitro and then re-introducing them into the nucleus is not possible, so the host organism has to be genetically engineered so that the fluorescent tag is an integral part of the protein that is synthesized in vivo. This is achieved by ligating the coding sequence for the green fluorescent protein (Tsien, 1998) to the gene for the protein being studied. Standard cloning techniques are then used to insert the modified gene into the host genome (Section 4.2.1), leading to a recombinant cell that synthesizes a fluorescent version of the protein. Observation of the cell using a fluorescence microscope now reveals the distribution of the labeled protein within the nucleus.

To study the mobility of the protein, a small area of the nucleus is photobleached by exposure to a tightly focused pulse from a high-energy laser. The laser pulse inactivates the fluorescent signal in the exposed area, leaving a region that appears bleached in the microscopic image. This bleached area gradually retrieves its fluorescent signal, not by a reversal of the bleaching effect, but by migration into the bleached region of fluorescent proteins from the unexposed area of the nucleus. Rapid reappearance of the fluorescent signal in the bleached area therefore indicates that the tagged proteins are highly mobile, whereas a slow recovery indicates that the proteins are relatively static. The kinetics of signal recovery can be used to test theoretical models of protein dynamism derived from biophysical parameters such as binding constants and flux rates (Misteli, 2001).

Image ch8fb1.jpg

From: Chapter 8, Accessing the Genome

Cover of Genomes
Genomes. 2nd edition.
Brown TA.
Oxford: Wiley-Liss; 2002.
Copyright © 2002, Garland Science.

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