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Uncoupling diffusion and binding in FRAP experiments

Nature Methods volume 6, page 183 (2009)Cite this article

Binding of fluorescent molecules to immobile sites will slow recovery after photobleaching provided the binding events are sufficiently frequent and long-lived. The effect of binding on FRAP depends on the relative rates of binding and diffusion. At the extreme where diffusion is much more rapid than binding, fluorescence recovery reflects only unbinding. These circumstances are referred to as diffusion-uncoupled, to distinguish them from instances in which fluorescence recovery is a complex function of both binding and diffusion (diffusion-coupled)3,4.

Schwarzenbacher et al.1 derive the lifetime of the CD4-Lck interaction after concluding that Lck contrast recovery is diffusion-uncoupled. They made this conclusion after comparing the time required for an Lck molecule to diffuse across a bleached region (the characteristic diffusion time) and the time required for recovery of fluorescence contrast. The diffusion time for full-length Lck (with a diffusion coefficient of ∼0.2–1.0 μm2 s−1) across 20 × 5 μm rectangles will be ∼6–30 s. The authors argue that this is sufficiently rapid compared to the 160 s required for recovery after photobleaching that diffusion is negligible and thus that the recovery rate corresponds to the unbinding rate. Although this makes intuitive sense, the diffusion-coupled and diffusion-uncoupled regimes cannot be distinguished simply by comparing diffusion and recovery rates4. A necessary preliminary step is to determine empirically whether diffusion has an impact on recovery. This is most commonly done by measuring recovery as a function of bleached region size.

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  1. Department of Pharmacology and Toxicology, Medical College of Georgia, 1120 15th St., Augusta, 30912, Georgia, USA

    Nevin A Lambert

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  1. Nevin A Lambert
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Lambert, N. Uncoupling diffusion and binding in FRAP experiments. Nat Methods 6, 183 (2009). https://doi.org/10.1038/nmeth0309-183a

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