Seeing Red

HeLa cell expressing CLONTECH's DsRed in mitochondria and Cyan Fluorescent Protein in the nucleus, visualized by overlaying fluorescence microscopy images Green fluorescent protein (GFP), isolated from the jellyfish Aequorea victoria, has been a valuable tool for researchers monitoring gene expression and protein distribution in living cells. Part of the appeal of using GFP to track gene expression is that it does not require addition of substrates or cofactors for detection, but is visualized

Aileen Constans
Jun 25, 2000


HeLa cell expressing CLONTECH's DsRed in mitochondria and Cyan Fluorescent Protein in the nucleus, visualized by overlaying fluorescence microscopy images
Green fluorescent protein (GFP), isolated from the jellyfish Aequorea victoria, has been a valuable tool for researchers monitoring gene expression and protein distribution in living cells. Part of the appeal of using GFP to track gene expression is that it does not require addition of substrates or cofactors for detection, but is visualized simply by applying UV or blue light radiation.1 Over the past few years, CLONTECH of Palo Alto, Calif., has expanded on this bright idea with its Living Colors family of fluorescent proteins. Reporters expressing proteins that glow in hues of blue, cyan, and yellow have been constructed by making GFP chromophore mutations that alter emission wavelength. Recent addition of the Living Colors DsRed Red Fluorescent Protein to this family makes simultaneous detection of multiple reporter proteins even more convenient.

Isolated from the Discosoma species of sea anemones, DsRed has an emission peak of 583 nm, which makes it compatible with standard rhodamine and propidium iodide filters. It can also be used with techniques such as flow cytometry and, since it is efficiently excited by 488 nm argon lasers, laser scanning confocal microscopy. But unlike GFP-based proteins, DsRed fluoresces outside the range of autofluorescence often observed from media, glassware, and cellular components. This feature leads to higher signal-to-noise ratios, enabling detection of low-abundance proteins. Also, DsRed fluoresces at a longer wavelength than do other autofluorescent proteins. Since tissues absorb more energy at shorter wavelengths, DsRed's fluorescence transmits more clearly through tissues, making it excellent for in vitro labeling of hard-to-label tissues.

According to market development manager Nicola Zahl, one of the main advantages of DsRed is that its excitation and emission peaks are very distinct from those of other Living Colors proteins. In the past, the only fluorescent proteins that could be combined in a dual-color labeling experiment were cyan and yellow; adding DsRed makes triple-labeling experiments possible.

CLONTECH currently offers four vectors for DsRed expression: pDsRed1-N1, for expression in mammalian tissue or creation of an N-terminal fusion protein; pDsRed1-C1, for creation of a C-terminal fusion protein; pDsRed, for expression in nonmammalian systems; and pDsRed1-1, for the study of promoter or enhancer elements. The protein has been expressed successfully in HEK-293, HeLa, Xenopus, and Escherichia coli cells.

For convenient detection of DsRed and DsRed fusion proteins on western blots, CLONTECH offers the Living Colors D.s. Peptide Antibody. In April the company introduced Mercury™ DsRed1 Signaling Probes, for detection of PKC or p53 activity in real time, and pDsRed1-Mito, a localization vector that targets mitochondria in living cells.

--Aileen Constans (aconstans@the-scientist.com)

1. M. Chalfie et al., "Green fluorescent protein as a marker for gene expression," Science, 263:802-5, 1994.

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