[Overview] [Experimental Setup] [Protocol] [Data Evaluation] [Examples] [Literature]

[How FRAP works] [Photobleaching] [Molecule Diffusion] [Recovery Dynamics] [Fluorophores]

< back     next >

For qualitative determination of the recovery dynamics, e.g. to compare differences of one molecule at different conditions, a simple exponential equation can be used as a first approximation:

After determination of t by fitting the above equation to the recovery curve the corresponding halftime of the recovery can be calculated with the following formula:

If the molecule binds to a slow or immobile macromolecular structure it is very likely that the recovery curve does not fit a single exponential equation. To overcome this problem, a biexponential equation can be used.

Depending on the investigated molecule the amount of interaction with other molecules will be variable. For example proteins which associate with relatively immobile cellular structures such as the cytoskeleton have a significantly reduced recovery compared to a freely mobile molecule.

Using kinetic modeling the binding characteristics of the examined molecule can determined by the ratio between mobile and immobile fraction.

< back     next >

An idealized plot of a FRAP recovery curve.

I_I: initial intensity
I₀: intensity at timepoint t₀ (first postbleach intensity)
I_1/2: half recovered intensity (I_1/2 = (I_E - I₀) / 2)
I_E: endvalue of the recovered intensity
t_half: Halftime of recovery corresponding to I_1/2 (t_1/2 - t₀) 
Mobile fraction F_m = (I_E - I₀) / (I_I - I₀)
Immobile fraction F_i = 1 - Fm

< back     next >

[EAMNET Home] [EAMNET News] [Introduction]

contact: Stefan Terjung           last update: 02/06/04