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Biochem J
2009 Mar 15;4183:567-74. doi: 10.1042/BJ20081949.
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Far-red fluorescent tags for protein imaging in living tissues.
Shcherbo D
,
Murphy CS
,
Ermakova GV
,
Solovieva EA
,
Chepurnykh TV
,
Shcheglov AS
,
Verkhusha VV
,
Pletnev VZ
,
Hazelwood KL
,
Roche PM
,
Lukyanov S
,
Zaraisky AG
,
Davidson MW
,
Chudakov DM
.
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A vast colour palette of monomeric fluorescent proteins has been developed to investigate protein localization, motility and interactions. However, low brightness has remained a problem in far-red variants, which hampers multicolour labelling and whole-body imaging techniques. In the present paper, we report mKate2, a monomeric far-red fluorescent protein that is almost 3-fold brighter than the previously reported mKate and is 10-fold brighter than mPlum. The high-brightness, far-red emission spectrum, excellent pH resistance and photostability, coupled with low toxicity demonstrated in transgenic Xenopus laevis embryos, make mKate2 a superior fluorescent tag for imaging in living tissues. We also report tdKatushka2, a tandem far-red tag that performs well in fusions, provides 4-fold brighter near-IR fluorescence compared with mRaspberry or mCherry, and is 20-fold brighter than mPlum. Together, monomeric mKate2 and pseudo-monomeric tdKatushka2 represent the next generation of extra-bright far-red fluorescent probes offering novel possibilities for fluorescent imaging of proteins in living cells and animals.
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