Recombinant Human TNF-alpha Hyperactive Mutant Protein

Recombinant Human TNF-alpha Hyperactive Mutant (Catalog # 8599-TA) induces cytotoxicity in the L-929 mouse fibroblast cell line in the presence of the metabolic inhibitor actinomycin D. The ED50 for this effect is 2.5-12.5 pg/mL. This is an approximate 10-fold activity increase over the wild-type TNF-alpha (Catalog # 210-TA).

Recombinant Human TNF-alpha Hyperactive Mutant (Catalog # 8599-TA) induces cytotoxicity in the L-929 mouse fibroblast cell line in the presence of the metabolic inhibitor actinomycin D. The ED50 for this effect is 2.5-12.5 pg/mL. This is an approximate 10-fold activity increase over the wild-type TNF-alpha (Catalog # 210-TA).

• Reactivity: Hu
• Applications: Bioactivity

Recombinant Human TNF-alpha Hyperactive Mutant Protein Summary

• Additional Information: Mutant form with approximately 5-fold greater activity
• Details of Functionality: Measured in a cytotoxicity assay using L‑929 mouse fibroblast cells in the presence of the metabolic inhibitor actinomycin D. Matthews, N. and M.L. Neale (1987) in Lymphokines and Interferons, A Practical Approach. Clemens, M.J. et al. (eds): IRL Press. 221. The ED₅₀ for this effect is 2.5-12.5 pg/mL.
• Source: E. coli-derived human TNF-alpha protein
  Lys87-Leu233 (Leu233Phe), with an N-terminal Met-Arg-Lys-Arg
• Accession #: P01375
• N-terminal Sequence: Met
• Structure / Form: Non-covalent trimer
• Protein/Peptide Type: Recombinant Proteins
• Gene: TNF
• Purity: >95%, by SDS-PAGE with silver staining.
• Endotoxin Note: <0.10 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

• Dilutions:
  • Bioactivity
• Theoretical MW: 17 kDa.
  Disclaimer note: The observed molecular weight of the protein may vary from the listed predicted molecular weight due to post translational modifications, post translation cleavages, relative charges, and other experimental factors.
• SDS-PAGE: 16 kDa, reducing conditions

Packaging, Storage & Formulations

• Storage: Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
  • 12 months from date of receipt, -20 to -70 °C as supplied.
  • 1 month, 2 to 8 °C under sterile conditions after reconstitution.
  • 3 months, -20 to -70 °C under sterile conditions after reconstitution.
• Buffer: Lyophilized from a 0.2 μm filtered solution in PBS with BSA as a carrier protein.
• Purity: >95%, by SDS-PAGE with silver staining.
• Reconstitution Instructions: Reconstitute at 100 μg/mL in PBS.

Notes

This product is produced by and ships from R&D Systems, Inc., a Bio-Techne brand.

Alternate Names for Recombinant Human TNF-alpha Hyperactive Mutant Protein

• APC1 protein
• Cachectin
• Cachetin
• DIF
• TNF
• TNF, monocyte-derived
• TNFA
• TNF-A
• TNFalpha
• TNF-alpha
• TNF-alphacachectin
• TNFATNF, macrophage-derived
• TNFG1F
• TNFSF1A
• TNFSF2
• TNFSF2TNF superfamily, member 2
• tumor necrosis factor (TNF superfamily, member 2)
• tumor necrosis factor alpha
• Tumor necrosis factor ligand superfamily member 2
• tumor necrosis factor
• tumor necrosis factor-alpha

Background

Tumor necrosis factor alpha (TNF-alpha ), also known as cachectin and TNFSF1A, is the prototypic ligand of the TNF superfamily. It is a pleiotropic molecule that plays a central role in inflammation, immune system development, apoptosis, and lipid metabolism (1, 2). Human TNF-alpha consisits of a 35 amino acid (aa) cytoplasmic domain, a 21 aa transmembrane segment, and a 177 aa extracellular domain (ECD) (3). Within the ECD, human TNF-alpha shares 97% aa sequence identity with rhesus and 71%-92% with bovine, canine, cotton rat, equine, feline, mouse, porcine, and rat TNF-alpha . TNF-alpha is produced by a wide variety of immune, epithelial, endothelial, and tumor cells (1, 2). TNF-alpha is assembled intracellularly to form a noncovalently linked homotrimer which is expressed on the cell surface (4). Cell surface TNF-alpha can induce the lysis of neighboring tumor cells and virus infected cells, and it can generate its own downstream cell signaling following ligation by soluble TNFR I (2, 5). Shedding of membrane bound TNF-alpha by TACE/ADAM17 releases the bioactive cytokine, a 55 kDa soluble trimer of the TNF-alpha extracellular domain (6-8). TNF-alpha binds the ubiquitous 55-60 kDa TNF RI (9, 10) and the hematopoietic cell-restricted 80 kDa TNF RII (11, 12), both of which are also expressed as homotrimers (1, 2, 13). Both type I and type II receptors bind TNF-alpha with comparable affinity (14), although only TNF RI contains a cytoplasmic death domain which triggers the activation of apoptosis. Soluble forms of both types of receptors are released and can neutralize the biological activity of TNF-alpha (15). This recombinant protein contains an N-terminal insertion and a Leu233Phe substitution which have been shown to increase the bioactivity of TNF-alpha in vitro and in vivo (16).

1. Zelova, H. and J. Hosek (2013) Inflamm. Res. 62:641.
2. Juhasz, K. et al. (2013) Expert Rev. Clin. Immunol. 9:335.
3. Pennica, D. et al. (1984) Nature 312:724.
4. Tang, P. et al. (1996) Biochemistry 35:8216.
5. Perez, C. et al. (1990) Cell 63:251.
6. Black, R.A. et al. (1997) Nature 385:729.
7. Moss, M.L. et al. (1997) Nature 385:733.
8. Gearing, A.J.H. et al. (1994) Nature 370:555.
9. Schall, T.J. et al. (1990) Cell 61:361.
10. Loetscher, H. et al. (1990) Cell 61:351.
11. Dembic, Z. et al. (1990) Cytokine 2:231.
12. Smith, C.A. et al. (1990) Science 248:1019.
13. Loetscher, H. et al. (1991) J. Biol. Chem. 266:18324.
14. Pinckard, J.K. et al. (1997) J. Biol. Chem. 272:10784.
15. Engelmann, H. et al. (1990) J. Biol. Chem. 265:1531.
16. Yan, Z. et al. (2006) Cytotherapy 8:415.

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FAQs for TNF-alpha (8599-TA). (Showing 1 - 4 of 4 FAQ).

1. I need to know about TNF-alpha antibody kits for mice.  ..show answer..
   • We currently have two TNFalpha ELISA kits specific for mouse: catalog numbers KA0257 and NBP1-92670.
2. I would like to ask you for help. I need an antibody for Elisa to detect human TNF alpha in a supernatant from a cell culture (meaning supernatant after centrifugation of collected cell suspension from a plate well). Which of your antibodies against human tnf alpha would be suitable? I would like to buy only the primary antibody.  ..show answer..
   • I would recommend catalog number NBP1-67821 or NB600-587; however, a full list of our anti-human TNF alpha antibodies suitable for use in ELISA can be found using this link.
3. I am interested in a TNF alpha antibody, cross reactive for human, rat and mouse (host species: rabbit). Could your product NBP1-19532 be used in western blotting applications? Or do you have a similar product in your catalog which could fit with my request?  ..show answer..
   • The antibody you mention, NBP1-19532, has not yet been validated in Western blot. I would instead recommend either NBP1-67821 or NB600-587. These are both rabbit polyclonal antibodies that cross-reacts with human, mouse and rat and have been used in Western blotting.
4. Can your TNF-alpha products be used to treat TBI victims and therefore avoid the perispinal injections with Enbrel?  ..show answer..
   • I am very sorry, but all of our products are for scientific research use only, and none are intended or approved for use in humans.

Bioinformatics

• Gene Symbol: TNF
• Uniprot:
  • P01375()