EnCor Biotechnology
Mouse Monoclonal Antibody to tdTomato Cat# MCA-6F12
Description
The MCA-6F12 antibody was made against full length recombinant tdTomato expressed in and purified from E. coli. The tdTomato protein contains two fluorescent Tomato units connected by a Ser and Gly rich linker sequence. This has superior spectral properties and is the form included in many widely used expression vectors. The antibody recognizes tdTomato strongly on western blots, in appropriate transfected and transgenic cells and tissues and does not react with GFP. The tdTomato protein was derived from DSRed and has a very strong orange fluorescence. The MCA-6F12 antibody also reacts with the closely related protein mCherry, also derived from DSRed. All these proteins are similar in size and general structure to GFP, though distinct in primary sequence and spectral properties (5,6).
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| Name: | tdTomato, mouse monoclonal, Cat# MCA-6F12 |
| Immunogen: | Full length two domain recombinant protein |
| UniProt: | D1MPT3 |
| Molecular Weight: | ~56kDa |
| Host: | Mouse |
| Isotype: | IgG1 |
| Format: | Affinity purified at 1mg/mL in PBS, 50% glycerol, 5mM NaN3 |
| Applications: | WB, IF/ICC, IHC |
| Recommended Dilutions: | WB: 1:2,000 IF/IHC: 1:1000-1:2,000. |
| Storage: | Stable at 4°C for one year, for longer term store at -20°C |
The Tomato fluorescent protein is derived from a natural product, DsRed, originally isolated as a red fluorescent protein from the coral of the genus Discosoma (1). Most vectors incorporating Tomato protein express two back to back copies, and this form is therefore referred to as "tandem dimer Tomato" or tdTomato. As with other natural fluorescent proteins of Cnidarians (jelly fish, sea anemones and corals), the natural form of the protein forms stable tetramers in vivo. DsRed, the original form, was engineered to improve its spectral properties and also prevent multimerization in the lab of Roger Tsien, where much work on fluorescent proteins was performed (2). Several further cycles of mutation, directed modification and evolutionary selection produced dTomato, which has an excitation maximum at 554nm and and emission maximum at 581nm (3). The protein is widely used as a fluorescent tracer in transfection, transgenic, photobleaching and FRET type experiments. The prototype for these fluorescent proteins is Green Fluorescent Protein (GFP), which is a ~27kDa protein isolated originally from the jellyfish Aequoria victoria (4). The Tomato protein is similar in size and general structural properties to GFP (5,6), but, obviously, produces a red rather than a green fluorochrome. As with GFP, Tomato becomes fluorescent due to intrinsic properties requiring only molecular oxygen and so can be readily expressed in a variety of systems.
This antibody was made against a recombinant tdTomato construct expressed in and purified from E. coli. The sequence is identical to that found in a series of widely used expression vectors, including two Tomato fluorescent units linked by a glycine and serine rich linker. The linker in the sequence below is underlined. The protein was expressed in the eukaryotic expression vector pET30a(+) which adds an N terminal His-tag and some other sequence, underlined below. This sequence includes a thrombin cleavage site (blue), an S-tag affinity peptide (red) and an enterokinase cleavage site (green).
MHHHHHHSSG LVPRGSGMKE TAAAKFERQH MDSPDLGTDD DDKAMADIGS EFMVSKGEEV
IKEFMRFKVR MEGSMNGHEF EIEGEGEGRP YEGTQTAKLK VTKGGPLPFA WDILSPQFMY
GSKAYVKHPA DIPDYKKLSF PEGFKWERVM NFEDGGLVTV TQDSSLQDGT LIYKVKMRGT
NFPPDGPVMQ KKTMGWEAST ERLYPRDGVL KGEIHQALKL KDGGHYLVEF KTIYMAKKPV
QLPGYYYVDT KLDITSHNED YTIVEQYERS EGRHHLFLGH GTGSTGSGSS GTASSEDNNM
AVIKEFMRFK VRMEGSMNGH EFEIEGEGEG RPYEGTQTAK LKVTKGGPLP FAWDILSPQF
MYGSKAYVKH PADIPDYKKL SFPEGFKWER VMNFEDGGLV TVTQDSSLQD GTLIYKVKMR
GTNFPPDGPV MQKKTMGWEA STERLYPRDG VLKGEIHQAL KLKDGGHYLV EFKTIYMAKK
PVQLPGYYYV DTKLDITSHN EDYTIVEQYE RSEGRHHLFL YGMDELYK
Number of amino acids: 528
Molecular weight: 59903.76
Theoretical pI: 6.08
Amino acid composition:
Ala (A) 21 4.0%
Arg (R) 20 3.8%
Asn (N) 10 1.9%
Asp (D) 33 6.2%
Cys (C) 0 0.0%
Gln (Q) 17 3.2%
Glu (E) 45 8.5%
Gly (G) 57 10.8%
His (H) 22 4.2%
Ile (I) 19 3.6%
Leu (L) 35 6.6%
Lys (K) 47 8.9%
Met (M) 25 4.7%
Phe (F) 24 4.5%
Pro (P) 28 5.3%
Ser (S) 30 5.7%
Thr (T) 31 5.9%
Trp (W) 6 1.1%
Tyr (Y) 28 5.3%
Val (V) 30 5.7%
Total number of negatively charged residues (Asp + Glu): 78
Total number of positively charged residues (Arg + Lys): 67
Extinction coefficients:
Extinction coefficients are in units of M-1 cm-1, at 280 nm measured in water.
Ext. coefficient 74720
Abs 0.1% (=1 g/l) 1.247
1. Matz MV, et al. Fluorescent proteins from nonbioluminescent Anthozoa species. Nat. Biotechnol. 17:969-73 (1999).
2. Baird GS, Zacharias DA, Tsien RY. Biochemistry, mutagenesis, and oligomerization of DsRed, a red fluorescent protein from coral. PNAS 97:11984-9 (2000).
3. Chalfie M, et al. Green fluorescent protein as a marker for gene expression. Science 263:802-5 (1994).
4. Gross LA. et al. The structure of the chromophore within DsRed, a red fluorescent protein from coral. PNAS 97:11990-5 (2000).
5. Heikal AA. et al. Molecular spectroscopy and dynamics of intrinsically fluorescent proteins: coral red (dsRed) and yellow (Citrine). PNAS 97:11996-2001 (2000).
6. Shaner NC. et al. Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein. Nat. Biotech. 22:1567-72 (2004).
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