Reversible fluorescent labeling with FAST
Applications
Reversible labeling, Silent Tag
FAST is a user-friendly, real-time fluorescent reporting system, which can act as a silent tag by non-permanent labeling with TFFluorogen.
A tag you can switch off
The FAST–fluorogen bond is non-covalent. Add the fluorogen and the tag lights up within seconds; remove it, a simple medium change, and the tag goes dark again, leaving the protein chemically untouched. Fluorescent proteins and covalent tags such as SNAP- or HaloTag mark a protein permanently; FAST, by contrast, gives you a signal that is entirely under your control, so a channel occupied one moment can be freed the next.
What reversibility lets you do
- Pulse–chase and turnover. Add fluorogen to mark a pool, wash it out, and follow what happens next without a lingering legacy signal.
- Longer imaging despite photobleaching. Because binding is dynamic, bleached fluorogen exchanges for fresh dye from the medium, sustaining signal over long acquisitions.
- Monitor transgenesis without a permanent mark. Check the efficiency of transgenesis in a cell line or organism, then silence the label, no permanently tagged line to carry forward.
- Free up detection channels. Silence one FAST report to release its channel for another probe, then restore it later.
- Switch colours live. The same reversibility underpins multicolour imaging with one tag, wash out one fluorogen, add another.
How reversibility works
Labelling is governed by a binding equilibrium, so the degree of labelling can be set by the fluorogen concentration and reversed by removing it. FAST gives strong fluorescence immediately on addition of the fluorogen, even in fully anaerobic conditions, and remains silent for as long as no fluorogen is in the medium.
Fluorogens for reversible labelling
Every FAST fluorogen labels reversibly, pick by colour from the products page, for example tfLime for green or tfCoral for orange-red. For surface-restricted, wash-controllable labelling, the non-permeant tfAmber-NP and tfFox-NP stay outside the cell.
Frequently asked
Is FAST labelling permanent?
No. Unlike fluorescent proteins or SNAP/HaloTag, FAST binds its fluorogen non-covalently, so the label is fully reversible.
How do I switch the signal off?
Remove the fluorogen from the medium with a wash or medium change; the tag falls silent and can be re-lit later by adding fluorogen again.
You are interested in
monitoring the efficiency of transgenesis without permanently labeling your transgenic cell line or organism?
Compatible techniques
MICROSCOPY
FLOW CYTOMETRY
MICROPLATE READERS
How can fast help you?
- FAST is a small purely monomeric protein, of only 14 kDa. Its small size and minimal genetic footprint (375 bp) minimize the risk of perturbation linked to the use of genetic tags.
- FAST binds non-covalently TFFluorogens, fluorogenic synthetic dyes that are dark in water and fluoresce only when bound to FAST, allowing specific detection of FAST.
- As TFFluorogen binds FAST non-covalently, labeling is non-permanent and can be easily reversed by washing TFFluorogens away.
Other key features:
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FAST provide strong fluorescence immediately upon addition of TFFluorogens even in fully anaerobic conditions.
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The level of labeling can be controlled by adjusting the concentrations of TFFluorogens.
- TFFluorogens come in a variety of colors: TFCoral (λem 600 nm), TFCitrus (λem 560 nm), TFAmber (λem 558 nm), TFLime (λem 540 nm).
recent paper about this application
A small fluorescence-activating and absorption-shifting tag for tunable protein imaging in vivo
Published in Proc. Natl. Acad. Sci. (USA) 113 (3), 497-502 (2016)
In this seminal paper, Pr. A. Gautier and L. Jullien evidence that fluorescent labeling with the FAST tag is highly dynamic and fully reversible...
…which strikingly contrasts with other tagging systems. As a consequence fluorescent tagging can be made transient, enabling for liberation of observation fluorescence channels. The FAST tag remains silent as long as no fluorogen is added to the medium.
Other Applications
Anaerobic imaging
Detecting protein-protein interactions
Protein & cell labeling
One tag, many colors
Reversible labeling, Silent Tag
A toolkit for sensing