| National Cancer Institute | Laboratory of Receptor Biology and Gene Expression | National Institutes of Health |
What are the exchange and diffusion rates of your protein of interest? How could you observe them in living cells?
| FRAP is a photobleaching technique that allows one to measure movement of molecules in real time. Fluorescent molecules in a target compartment are bleached with a brief laser beam pulse of the appropriate wavelength. Subsequent observation of the target compartment will determine if the molecules are fixed (compartment remains bleached) or mobile (compartment refills with new fluorescent molecules). Rates of movement can be determined from a quantitative evaluation of the data. |
| In FLIP, the photobleaching laser is directed at fluorescent molecules away from the target, and bleaching is carried out in a pulsed mode over a longer period of time. As the pool of labeled molecules is depleted, behavior of the target structure is observed. An advantage of this approach is that neither the target structure, nor the fluorescent molecules at the target, are subjected to radiation. |
Dr. Gordon Hager (Chief, Lab of Receptor Biology and Gene Expression) studies the mechanism of glucocorticoid receptor gene activation. His research is an example of how microscopy can be used to assay exchange rates of proteins at binding targets in vivo.
Binding of the glucocorticoid receptor (GR) to the mouse mammary tumor virus promoter (MMTV) has been used as a model system to study the mechanism of transcription. It has been proposed that the receptor may interact transiently with a response element, recruiting a secondary set of factors that in turn form a stable complex at the regulatory site (a "hit and run" mechanism). To test this, a cell line containing 200 copies of the MMTV promoter in a tandem array was examined. These cells also contained a GFP-tagged GR, enabling visualization of GFP-GR binding to the large tandem array. To examine GR exchange rates on the DNA, Hager and colleagues used FRAP (fluorescence recovery after photobleaching). In FRAP, a laser operating at high power bleaches a region of interest, and then that region is monitored for recovery of fluorescence. The rate of recovery reflects the rate of protein exchange at that site. Exchange at the GFP-GR array occurred within seconds, consistent with a "hit and run" mechanism (Figure 4A-F). To verify that the observed rapid exchange was not an artifact due to bleaching of bound GFP-GR molecules, Hager and colleagues performed FLIP (fluorescence loss in photobleaching). In FLIP, a laser is used to repeatedly bleach molecules not at the target in order to gradually deplete the total amount of available fluorescence, without directly bleaching molecules at the target. Using FLIP, Hager and colleagues found that fluorescence at the array gradually disappeared (Figure 4G-N). This implies that the unbleached (and undamaged) GFP-GR molecules present at the array were still exchanging.