Recombinant TEV Protease Protein, CF Summary
Accession # NP_062908
CF stands for Carrier Free (CF). We typically add Bovine Serum Albumin (BSA) as a carrier protein to our recombinant proteins. Adding a carrier protein enhances protein stability, increases shelf-life, and allows the recombinant protein to be stored at a more dilute concentration. The carrier free version does not contain BSA.
In general, we advise purchasing the recombinant protein with BSA for use in cell or tissue culture, or as an ELISA standard. In contrast, the carrier free protein is recommended for applications, in which the presence of BSA could interfere.
|Formulation||Supplied as a 0.2 μm filtered solution in Tris, NaCl, EDTA, DTT and Glycerol.|
|Shipping||The product is shipped with dry ice or equivalent. Upon receipt, store it immediately at the temperature recommended below.|
|Stability & Storage:||Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
- Assay Buffer: 50 mM Tris, 0.5 mM EDTA, 1 mM DTT, pH 8.0
- Recombinant Viral TEV Protease (rvTEV) (Catalog # 4469-TP)
- Substrate: Any fusion protein containing the recognition sequence ENLYFQ. The cleavage site is after glutamine (Q).
- SDS-PAGE followed by protein staining
- Dilute rvTEV Protease to 0.02 mg/mL in Assay Buffer.
- Dilute Substrate to 0.5 mg/mL in Assay Buffer.
- Form reaction mixture by combining 20 µL of diluted Substrate and 20 µL Assay Buffer. Also prepare two additional mixtures, to be used as controls, containing 20 µL of diluted Substrate and 30 µL of Assay Buffer.
- Incubate all three mixtures (temperature equilibration), as well as the diluted rvTEV Protease, for 15 minutes at room temperature.
- Add 10 µL diluted rvTEV Protease to the reaction mixture, excluding the controls, for a final volume of 50 µL in each reaction.
- Incubate the reaction containing the diluted rvTEV Protease as well as one of the controls for 1 hour at room temperature. Keep the other control on ice.
- Stop the reactions by mixing equal volumes of reaction mixture (including controls) and 2X reducing SDS-PAGE sample buffer together. Heat for 5 minutes at 100 °C.
- Analyze the cleavage by SDS-PAGE followed by protein staining.
- rvTEV Protease: 0.2 µg
- Substrate: 10 µg
Background: TEV Protease
TEV Protease is the 241 amino acid (aa), 27 kDa catalytic domain of the nuclear inclusion a (NIa) protein encoded by the potyvirus, tobacco etch virus (TEV). It may be used in biotechnology to cleave affinity tags from recombinant proteins, either co-translationally or in vitro following purification. Its high specificity and activity at a wide range of pH and ionic strength make TEV Protease more versatile than many other proteases used for the same purpose. Unlike factor Xa, enteropeptidase or thrombin, TEV Protease has not been found to cleave at unintended sites, even when present at a high concentration. TEV Protease is a 3C-type protease that cleaves substrates with a consensus sequence of ENLYFQG. Cleavage occurs between Q and G. Since the final aa remains on the cleaved protein where it could potentially affect structure or function, substitution of a variety of aa have been tested. In order of efficiency, S, A, M, Y, D, N, E, K or L may be effectively used in place of G. Several of the remaining aa may also vary, giving a final consensus sequence of ExxYF(M)Q(E)/G(S, A or others) where aa in parenthesis are alternatives and x is any aa. The autocatalytic site of NIa at S2256 has been mutated to an N for improved stability of the protease.
- Daros, J.-A. et al. (1999) J. Virol. 73:8732.
- Mondigler, M. and M. Ehrmann (1996) J. Bacteriol. 178:2986.
- Phan, J. et al. (2002) J. Biol. Chem. 277:50564.
- Kapust, R.B. et al. (2002) Biochem. Biophys. Res. Commun. 294:949.
- Kapust, R.B. et al. (2001) Protein Eng. 14:993.
Citations for Recombinant TEV Protease Protein, CF
R&D Systems personnel manually curate a database that contains references using R&D Systems products. The data collected includes not only links to publications in PubMed, but also provides information about sample types, species, and experimental conditions.
Citations: Showing 1 - 2
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Acoustic biosensors for ultrasound imaging of enzyme activity
Authors: A Lakshmanan, Z Jin, SP Nety, DP Sawyer, A Lee-Gossel, D Malounda, MB Swift, D Maresca, MG Shapiro
Nat. Chem. Biol., 2020;0(0):.
Applications: Enzyme Assay
The Vesicle Priming Factor CAPS Functions as a Homodimer via C2 Domain Interactions to Promote Regulated Vesicle Exocytosis
J Biol Chem, 2016;0(0):.
Sample Types: Recombinant Protein
Applications: Enzyme Assay
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