FLIP-FRAP

In this section we will discuss various FRAP variants that have been used to study the behaviour of nuclear proteins. The development of confocal microscopy in the 1980s made it possible to extend the straightforward methods provided by early researchers. The scanning capacity of this type of microscopes made it possible to bleach specific areas and measure the redistribution of fluorescence in other regions, or even scan the entire cell. This enabled various more dedicated approaches that will be discussed below FRAP-FIM (FRAP for immobilisation measurement) (Fig. 3A) [ 11], combined strip-FRAP (Fig. 3B) and FLIP-FRAP (Fig. 3C,E) [22], FLIP (Fig. 3D) [28-30, 32-34] and iFRAP (Fig. 3F) [25]. 

When binding times are very short, typically less than approximately 5 s, the combined strip-FRAP/FLIP-FRAP method, or any other FRAP-method is not capable to distinguish this scenario from a situation where aU molecules are freely mobile (but moving slower). However, several approaches to cope with this problem have been developed. In the investigation of nuclear factors, it can... 

Fig. 4A-H Comparison of two different variants of FRAP strip-FRAP and FLIP-FRAP (see also Fig. 3B, C). The combined use of the two protocols may allow to discriminate between transient binding and slow diffusion. The curves are based on computer simulated FRAP experiments (see the text) A, B schematic drawings of the strip-FRAP (see also Fig. 1C) and FLIP-FRAP methods. The FLIP-FRAP method differs from the strip-FRAP in that two areas are monitored after bleaching. Briefly, a strip at one pole of the nucleus is bleached for a relatively long period at a moderate excitation intensity. Subsequently the fluorescence is monitored in that region (FRAP), but also in the area at the other side of the nucleus (FLIP). Subsequently the difference between the two (normalised) fluorescence levels is plotted against time C schematic drawing of two scenarios where molecules are either free, but relatively slow (D=4 pmVs, top panel), or relatively fast (D=7 pm /s), but transiently immobilised such that 30% is immobile in steady state and individual molecules are immobilised for 45 s (bottom panel) D, E strip-FRAP and FLIP-FRAP curves of the scenarios depicted in C. In this case strip-FRAP can discriminate between the two cases, whereas the FLIP-FRAP curves are nearly identical F schematic drawing of a situation where freely mobile molecules are slower (D=l pmVs, top panel) than in C G,H strip-FRAP curves are identical whereas the FLIP-FRAP method can now discriminate between the two scenarios...

Rabut, Q., and Ellenberg, J. (2005). Photobleaching Techniques to Study Mobility and Molecular Dynamics ofProteins in Living Cells FRAP, iFRAP, and FLIP. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 

The observation was that bleached GFP-GR is rapidly replaced with unbleached GFP-GR (FRAP) (Fig. 7), and if GFP-GR is bleached elsewhere in the nucleus, GFP-GR is replaced with non-fluorescent GR molecules (either bleached GFP-GR or endogenous unlabelled GR) on the array (FLIP). The conclusion from these observations is that GFP-GR exchanges at a high rate between the array promoters and the nucleoplasm. Observations made in live cells has led to a completely... 

Fig. 7. GFP-GR bound to the MMTV array was analyzed by fluorescence recovery after photobleaching (FRAP). The bleached region is indicated in the image of the pre-bleached nucleus (A). The pre-bleach array is shown in (B), the post-bleach image (C), and at 4.1 s (D) and 11.6 s (E) post-bleach. This analysis, along with Fluorescence Loss in Photobleaching (FLIP) experiments, show that GFP-GR undergoes rapid exchange with the array (from Ref. [58]). Scale bar 5 pm.

Examples of useful applications of confocal FRAP include selective photobleaching and fluorescence loss in photobleaching (FLIP) (18, 55). In contrast with conventional spot photobleaching FRAP, in confocal FRAP it possible to visualize both the bleach region and the surrounding area of the cell. In addition, confocal FRAP techniques typically use... 

Ras trafficking to cellular membranes can be measured by fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP) (54). Both techniques rely on the expression of fluorescent-labeled Ras proteins to monitor different parameters of Ras movement across and between cellular membranes. FRAP involves photobleaching a membrane subdomain and measuring the kinetics of fluorescence recovery—and hence Ras trafflcking—into the bleached area. With FLIP, a cellular membrane is photobleached repeatedly and the subsequent intercellular movement of the photobleached area is monitored. 

In another approach, the interfacial diffusion of the nanoparticles was determined using two photobleaching methods fluorescence loss induced by photobleaching (FLIP) and fluorescence recovery after photobleaching (FRAP). It was found that the lateral diffusion of the nanoparticles at the interface as well as the diffusion normal to and from the interface deviated by about four orders of magnitude from the values obtained in free solution [46],... 

AR Androgen receptor ER Estrogen receptor FLIP Fluorescence loss in photobleaching FRAP Fluorescence recovery after photobleaching FRET Fluorescence resonance energy transfer GR Glucocorticoid receptor NER Nucleotide excision repair UV Ultra-violet... 

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