The membrane non-permeant fluorogen TFFox-NP adds to the ever-expanding toolkit of The Twinkle Factory.  Indeed, it is coming after the dark chromophore TFDarth just released, and the far-red fluorogen TFPoppy in 2020.

TFFox-NP belongs to the group of membrane non-permeant fluorogens.  While they have a reduced cellular uptake due to their chemical structure, they allow the specific labeling of FAST-tagged proteins located at the cell surface.  Used in combination with permeant fluorogens, they allow to access to both populations of inner and surface proteins.  As a result, powerful yet simple protocols based on those allow high throughput screening.  For example, one can evaluate the ability of small molecules to rescue trafficking defects by simple flow cytometry, with no need to image.  This hence opens great prospects for the discovery of drugs treating trafficking-related diseases.

Besides, compatible with STED, FRET, etc.  And, as always with FAST, works in anaerobic conditions!


The science around TFFox-NP

painting with FAST

painting with FAST – © The Authors 2021

TFFox-NP originates from the research led by Prof. Arnaud Gautier at Sorbonne University.  He gathered colleagues from Collège de France and ENS of PSL Research University, Institute of Psychiatry and Neuroscience of Paris, etc.  As a result of their collaboration, they issued an insightful paper in Nat. Commun. 2021.

The novel promiscuous variant therein disclosed, pFAST, offers optimized chromophore binding affinity and fluorescence brightness.  As a result, it allows tuning the fluorescence colors from blue to red, in the most demanding setups, e.g., FRET, STED.  This paper also discloses a series of new chromophores optimized for pFAST.  Of those, the non-permeant HBRAA-3,5DM.

The authors disclose that pFAST efficiently interacts with the unreported HBRAA-3,5DM, forming a bright orange fluorescent assembly HBRAA-3,5DM.  pFAST:HBRAA-3,5DM is indeed 2.5 to 4 times brighter than FAST:HBRAA-3E, the most efficient combination for selective labeling of cell surface proteins reported so far.


TFFox-NP and pFAST plasmids now available

The Twinkle Factory now distributes HBRAA-3,5DM with the tradename TFFox-NP under the reference # 524578-250.

We are happy to apply a two-for-the-price-of-one discounts for first orders of TFFox-NP!

Luc Lenglet, C.E.O. Twinkle Bioscience

As usual, one can order published pFAST plasmids from Addgene.  For bespoke plasmids, ask e-Zyvec.


More reading on non-permeant fluorogens

  • Engineering of a fluorescent chemogenetic reporter with tunable color for advanced live-cell imaging, Nat. Commun. 2021: the recent disclosure of optimized membrane non-permeant fluorogens for pFAST, e.g., HBRAA-3,5DM
  • Visualizing the dynamics of exported bacterial proteins with the chemogenetic fluorescent reporter FAST, Sci. Rep. 2020: first utilization of membrane non-permeant fluorogens with FAST in both monoderm and diderm bacteria, resp., L. monocytogenes and E. coli
  • Fluorogenic Probing of Membrane Protein Trafficking. Bioconjug. Chem. 2018: the introduction of the first-ever membrane non-permeant fluorogens for FAST, namely, HBRAA-3E, a.k.a. tfAmber-NP