RRC ID 50519
Author Kogure T, Karasawa S, Araki T, Saito K, Kinjo M, Miyawaki A.
Title A fluorescent variant of a protein from the stony coral Montipora facilitates dual-color single-laser fluorescence cross-correlation spectroscopy.
Journal Nat Biotechnol
Abstract Dual-color fluorescence cross-correlation spectroscopy (FCCS) is a promising technique for quantifying protein-protein interactions. In this technique, two different fluorescent labels are excited and detected simultaneously within a common measurement volume. Difficulties in aligning two laser lines and emission crossover between the two fluorophores, however, make this technique complex. To overcome these limitations, we developed a fluorescent protein with a large Stokes shift. This protein, named Keima, absorbs and emits light maximally at 440 nm and 620 nm, respectively. Combining a monomeric version of Keima with cyan fluorescent protein allowed dual-color FCCS with a single 458-nm laser line and complete separation of the fluorescent protein emissions. This FCCS approach enabled sensitive detection of proteolysis by caspase-3 and the association of calmodulin with calmodulin-dependent enzymes. In addition, Keima and a spectral variant that emits maximally at 570 nm might facilitate simultaneous multicolor imaging with single-wavelength excitation.
Volume 24(5)
Pages 577-81
Published 2006-5-1
DOI 10.1038/nbt1207
PII nbt1207
PMID 16648840
IF 36.553
Times Cited 210
Resource
DNA material tdKeima/pRSETB (RDB15232) mKeima/pcDNA3 (RDB15233) dKeima/pRSETB (RDB15234) dKeima570/pRSETB (RDB15235) CoralHue™ humanized monomeric Keima-Red (phmKeima-Red-MNL) (RDB18279) CoralHue™ monomeric Keima-Red (pmKeima-Red-S1) (RDB18280) CoralHue™ monomeric Keima-Red (pmKeima-Red-MN1) (RDB18281) CoralHue™ humanized monomeric Keima-Red (phmKeima-Red-S1) (RDB18282) CoralHue™ humanized monomeric Keima-Red (phmKeima-Red-MCL) (RDB18283) CoralHue™ humanized dimeric Keima-Red (phdKeima-Red-MNL) (RDB18284) CoralHue™ dimeric Keima-Red (pdKeima-Red-S1) (RDB18285) CoralHue™ humanized dimeric Keima-Red (phdKeima-Red-S1) (RDB18286) CoralHue™ humanized dimeric Keima-Red (phdKeima-Red-MCL) (RDB18287) CoralHue™ humanized dimeric Keima570 (phdKeima570-MNL) (RDB18293) CoralHue™ dimeric Keima570 (pdKeima570-S1) (RDB18294) CoralHue™ humanized dimeric Keima570 (phdKeima570-S1) (RDB18295) CoralHue™ humanized dimeric Keima570 (phdKeima570-MCL) (RDB18296) CoralHue™ Mitochondria-targeted mKeima-Red (pMT-mKeima-Red) (RDB18321) CoralHue™ Plasma Membrane-targeted mKeima-Red (pPM-mKeima-Red) (RDB18322) CoralHue™ Mitochondria-targeted monomeric Midoriishi-Cyan 1 (RDB18323) CoralHue™ Nucleoplasm-targeted humanized dimeric Keima-Red (RDB18324) CoralHue™ Nucleoplasm-targeted humanized Kusabira-Cyan 1 (pNP-hKCy1) (RDB18325) CoralHue™ Nucleoplasm-targeted humanized dimeric Keima570 (RDB18327)