Far-red fluorescent tags for protein imaging in living tissues

Biochem J. 2009 Mar 15;418(3):567-74. doi: 10.1042/BJ20081949.

Abstract

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Cell Line
  • Diagnostic Imaging / methods
  • Embryo, Nonmammalian
  • Fluorescence
  • Fluorescent Dyes* / chemistry
  • HeLa Cells
  • Humans
  • Luminescent Proteins* / chemistry
  • Luminescent Proteins* / genetics
  • Molecular Sequence Data
  • Sequence Alignment
  • Xenopus laevis

Substances

  • Fluorescent Dyes
  • Luminescent Proteins
  • red fluorescent protein