Far-red fluorescent protein Katushka2S

- Super bright far-red fluorescence
- High signal-to-noise ratio, fast maturation
- Recommended for whole body imaging

Katushka2S is the next generation of far-red fluorescent protein TurboFP635 (Katushka) [Luker et al., 2015; Shcherbo et al., 2009; Gurskaya et al., 2011]. Katushka2S exhibits highest brightness, fastest maturation and superior signal-to-noise ratio compared to TurboFP635 and many other available far-red fluorescent proteins: E2-Crimson, mNeptune, mNeptune2.5, mCardinal, TurboFP650 and NirFP [Luker et al., 2015], which makes it the protein of choice for visualization within living tissues. Katushka2S can also be used together with iRFP720 [Luker et al., 2015] for dual color whole body imaging.

Main properties

Katushka2S normalized excitation (thin line) and emission (thick line) spectra. Spectra viewer tool Download Katushka2S spectra (xls)

CHARACTERISTIC * Brightness is a product of extinction coefficient and quantum yield, divided by 1000. Molecular weight, kDa 26 Polypeptide length, aa 235 Fluorescence color far-red Excitation maximum, nm 588 Emission maximum, nm 633 Quantum yield 0.44 Extinction coefficient, M-1cm-1 67 000 Brightness* 29.5 Brightness, % of EGFP 89 pKa 5.4 Structure dimer Aggregation no Maturation rate at 37°C super fast Photostability,widefield (sec) 72 Cell toxicity not observed Main advantages super bright and fast-maturing far-red fluorescent protein; high pH-stability Possible limitations dimer, limited applicability for fusions generation, medium photostability

Recommended filter sets and antibodies

Katushka2S can be recognized using Anti-tRFP antibody (Cat.# AB233) available from Evrogen.

Recommended Omega Optical filter sets are QMAX-Red and XF102-2. Katushka2S can also be detected using Texas Red filter sets or similar.
For IVIS Lumina II imaging system the highest fluorescence signal for Katushka2S is observed with the following settings:
In cell culture: "Cy5.5" (695-770 nm) channel using 570/35nm excitation
In whole body imaging: "Cy5.5" (695-770 nm) channel using 605/35nm excitation

Performance and use

Mammalian cells transiently transfected with Katushka2S expression vectors produce bright fluorescence in 12 hrs after transfection. No cytotoxic effects or visible protein aggregation are observed.

Superior performance of Katushka2S in whole-body imaging was demonstrated using mouse xenograft model. HEK293FT cells transiently transfected with plasmids encoding different far-red fluorescent proteins were implanted into mice intramuscularly or subcutaneously. The cells were co-transfected with firefly luciferase to normalize the transfection efficiency and total numbers of injected cells. Katushka2S produced higher fluorescence signal and better signal-to-noise ratio than other far-red fluorescence proteins analyzed at various excitation and emission channels.

Katushka2S was also compared with one of the best phytochrome photoreceptors iRFP720. Katushka2S produced brighter fluorescence intensity at its optimum conditions as compared with the best output from iRFP720. At the same time, both proteins showed comparable signal-to-noise ratios at the optimum wavelengths for excitation and emission since autofluorescence from mouse tissue was lower in the channel optimal for iRFP720.

Note: Katushka2S demonstrates lower photostability than its parental variant TurboFP635. It may limit Katushka2S utility in cell culture experiments, but not in whole body imaging, where scattering and absorption of light naturally limit excitation power [Leblond et al., 2010].

Available variants and fusions

Variant	Description	Related vector	Cat.#
Humanized Katushka2S	Katushka2S codon usage is optimized for high expression in mammalian cells [Haas et al., 1996], but it can be successfully expressed in many other heterological systems.	pKatushka2S-C	FP761
		pKatushka2S-B	FP763