td-Tomato

Target Background

td-Tomato is derived from proteins originally isolated from Cnidarians (jelly fish, sea anemones and corals), and is used as a fluorescent tracer in trasfection and transgenic experiments. The prototype for these fluorescent proteins is Green Fluorescent Protein (GFP), which is a ~27 kDa protein isolated originally from the jellyfish Aequoria victoria. GFP was the basis of the 2008 Nobel Prize in Chemistry, awarded to Osamu Shimomura, Martin Chalfie and Roger Tsien, specifically “for the discovery and development of the green fluorescent protein, GFP”. GFP was shown to fluoresce on contact with molecular oxygen, requiring no other cofactors, and so can be expressed in fluorescent form in essentially any prokaryotic or eukaryotic cell. The td-Tomato protein is derived from DsRed, a red fluorescent protein related to GFP isolated from so-called disc corals of the genus Discosoma. DsRed is similar in size and properties to GFP, but, obviously, produces a red rather than a green fluorochrome. The original DsRed was engineered extensively in the Tsien lab to prevent it from forming tetramers and dimers and to modify and improve the spectral properties (1-3). Several further cycles of mutation, directed modification and evolutionary selection produced td-Tomato, which has an excitation maximum at 587 nm and and emission maximum at 610 nm (4).We expressed the td-Tomato protein sequence shown in reference 4 in bacteria, purified out the td-Tomato and raised this rabbit polyclonal antibody. This was affinity purified and was found to stain a band of the expected size in HEK293 cells transfected with the pFin-EF1-td-Tomato vector designed to express td-Tomato which was obtained from Clontech. As shown below, the antibody does not stain any protein band in untransfected HEK293 cells.