FRAP Analysis Calculator

Calculate diffusion coefficient and mobile fraction from Fluorescence Recovery After Photobleaching data.

Half-recovery time t½ (seconds) (s)

Bleach spot radius r (μm) (μm)

Fluorescence at t=0 before bleach F0 (AU)

Fluorescence immediately after bleach Fb (AU)

Fluorescence at plateau (recovery) Finf (AU)

Please check your inputs and try again.

Two Independent Readouts

A FRAP curve yields two separate quantities, and they answer different biological questions.

Measure	From	Reports
Diffusion coefficient	Recovery half-time	How fast mobile molecules move
Mobile fraction	Plateau height	What proportion can move at all

D = 0.224 r² / t_½ Mobile % = (F_∞ − F₀)/(F_pre − F₀) × 100

An incomplete plateau means some population is genuinely immobile — anchored to cytoskeleton, incorporated into a stable complex, or otherwise tethered. A protein can diffuse rapidly yet have a large immobile fraction, and reporting only D would miss that entirely.

Molecule	D (μm²/s)
Soluble GFP in cytoplasm	20–30
Cytoplasmic protein complex	1–10
Membrane protein	0.05–0.5
Membrane lipid	0.5–5
Chromatin-bound factor	< 0.1, often largely immobile

Membrane proteins diffuse roughly a hundredfold slower than cytoplasmic ones, because the lipid bilayer is far more viscous than cytosol and many are tethered to underlying cytoskeleton.

Common Pitfalls

Problem	Consequence	Handling
Photobleaching during recovery	Underestimates mobile fraction	Minimise imaging laser power
Bleach spot too large	Recovery slow, D underestimated	Match spot to expected mobility
Cell movement or drift	Distorts the curve	Correct with a reference region
Binding as well as diffusion	D is apparent, not true	Vary spot size to test

The binding issue is fundamental. If a protein binds transiently while diffusing, recovery reflects both processes and the extracted D is an effective value. Varying the bleach spot radius distinguishes them: pure diffusion scales t_½ with r², while binding-dominated recovery does not.

Worked Examples

Example 1: Membrane protein: t½=5s, r=2μm

D=0.224×4/5=0.179μm²/s

Result: Slow lateral diffusion — membrane protein

Transmembrane proteins: 0.01-0.1μm²/s

Example 2: GFP in cytoplasm: t½=0.5s, r=2μm

D=0.224×4/0.5=1.79μm²/s

Result: Faster diffusion in cytoplasm

Free GFP: 20-30μm²/s, bound GFP: slower

Example 3: Fast but immobile

D = 15 μm²/s, mobile fraction 35%

Result: Both readouts needed

The mobile population moves quickly, but nearly two thirds cannot move at all. Reporting only D would suggest high mobility.

Example 4: Testing for binding

Double the bleach radius

Result: t_½ should quadruple for pure diffusion

If recovery time scales less than r², binding contributes and the extracted D is an effective rather than true diffusion coefficient.

Common Mistakes

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Reporting D without the mobile fraction

They are independent. A protein can diffuse quickly while most of the population is immobilised, and D alone would not reveal that.

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Ignoring photobleaching during the recovery phase

Imaging itself bleaches, flattening the plateau and understating the mobile fraction. Use the lowest workable laser power.

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Treating apparent D as pure diffusion

Transient binding contributes to recovery. Varying bleach spot radius distinguishes them, since only true diffusion scales t_½ with r².

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Comparing D across different spot sizes

The formula includes r², so spot geometry must be reported. Values from different setups are not directly comparable.

Frequently Asked Questions

Mobile vs immobile fraction?

If 30% of GFP doesn't recover (IF=30%): protein is bound to immobile structure (cytoskeleton, DNA) or in membrane raft. Mobile fraction = freely diffusing molecules. FRAP measures combination of diffusion AND binding reactions.

FRAP vs FCS?

FRAP: bleach region, measure recovery time. FCS (Fluorescence Correlation Spectroscopy): analyze fluctuations of single molecules passing through small volume. FCS: better for fast diffusion, single-molecule sensitivity. FRAP: better for slow diffusion, large populations, in vivo.

What does the mobile fraction tell you?

The proportion of molecules free to move. An incomplete recovery plateau indicates a population anchored to cytoskeleton or held in a stable complex.

Why do membrane proteins diffuse so slowly?

The lipid bilayer is far more viscous than cytosol, and many membrane proteins are tethered to underlying cytoskeleton. Values are roughly a hundredfold lower.

How do I know whether binding contributes?

Vary the bleach spot radius. Pure diffusion makes recovery time scale with r²; binding-dominated recovery does not.

Why does imaging affect the result?

The imaging laser bleaches fluorophores during recovery, flattening the plateau and understating the mobile fraction. Use minimal power.