Recombinant Pro-TNF-alpha Fusion Protein, CF

• Reactivity: Hu
• Applications: Enzyme Activity
• Format: Carrier-Free

Recombinant Pro-TNF-alpha Fusion Protein, CF Summary

• Details of Functionality: Measured by its ability to be used as a protein substrate for TACE/ADAM17. Under the described conditions TACE/ADAM17 will cleave pro-TNF-alpha to produce mature TNF-alpha .
• Source: E. coli-derived human TNF-alpha protein
  

  Bacterial Protein Fusion Partner	Human Pro-TNF-alpha (Gly57-Ala76) Accession # P01375	Human Mature TNF-alpha (Val77-Leu233) Accession # P01375
  N-terminus		C-terminus

• Accession #: P01375
• N-terminal Sequence: Bacterial Protein Fusion Partner
• Protein/Peptide Type: Recombinant Proteins
• Gene: TNF
• Purity: >90%, by SDS-PAGE under reducing conditions and visualized by silver stain.
• Endotoxin Note: <1.0 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

• Dilutions:
  • Enzyme Activity
• Theoretical MW: 45 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: 42 kDa, reducing conditions

Packaging, Storage & Formulations

• Storage: Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
  • 6 months from date of receipt, -20 to -70 °C as supplied.
  • 3 months, -20 to -70 °C under sterile conditions after reconstitution.
• Buffer: Lyophilized from a 0.2 μm filtered solution in Urea, NaCl, NaH₂PO₄ and DTT.
• Purity: >90%, by SDS-PAGE under reducing conditions and visualized by silver stain.
• Reconstitution Instructions: Reconstitute at 0.2 mg/mL in sterile, deionized water.
• Assay Procedure:
  • Assay Buffer: 25 mM Tris, 200 mM NaCl and 0.02%(w/v) Brij-35, pH 8.0
  • Recombinant Human Pro TNF‑ alpha Fusion Protein (rhPro-TNF-alpha ) (Catalog # 1012-PS)
  • Recombinant Human TACE/ADAM17 (rhTACE) (Catalog # 930-ADB)
  • Positive Control: Recombinant Human TNF-alpha (rhTNF-alpha ) (Catalog # 210-TA)
  • Goat Anti-Human TNF‑ alpha Biotinylated Antigen Affinity-purified Polyclonal Antibody (Catalog # BAF210)
  • SDS-PAGE followed by Western Blotting
  1. Prepare the following vials for a final volume of 20 µL.
  1. 10 µL of rhPro-TNF-alpha at 0.2 mg/mL + 10 µL of rhTACE at 0.2 mg/mL
  2. 10 µL of rhPro-TNF-alpha at 0.2 mg/mL + 10 µL of Assay Buffer (control-with incubation)
  3. 10 µL of rhPro-TNF-alpha at 0.2 mg/mL + 10 µL of Assay Buffer (control-without incubation)
  2. Incubate vials at 37 °C overnight (except for the non-incubated control, store at ≤-20 °C).
  3. Stop the reactions and controls by adding the reducing gel loading buffer for SDS-PAGE to all vials for a final concentration of 50 ng rhPro-TNF-alpha /15 µL. Heat the samples at 95 °C for 3-5 minutes.
  4. Prepare a sample of rhTNF-alpha (Catalog # 210-TA) at 5 ng/15 µL in reducing gel loading buffer as a control on the gel. Heat the sample at 95 °C for 3-5 minutes.
  5. Load the samples on a 15% gel.
  1. 50 ng/lane (15 µL) of rhPro-TNF-alpha of the incubated reactions (including the control with incubation).
  2. 50 ng/lane (15 µL) of rhPro-TNF-alpha of the control (without incubation).
  3. 5 ng/lane (15 µL) of rhTNF-alpha (Catalog # 210-TA) control.
  6. Follow SDS-PAGE/Western blotting procedures.
  1. Use the Biotinylated Anti-human TNF-alpha /TNFSF1A antibody at 0.1 μg/mL. (Catalog # AF-210-NA) antibody may be used in place of (Catalog # BAF210).
  7. Visually determine processing of rhPro-TNF-alpha to the mature form by rhTACE by comparing the incubated reactions to the rhTNF-alpha control.

Notes

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

Alternate Names for Recombinant Pro-TNF-alpha Fusion Protein, CF

• 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 TNFSF2, 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 TNF-alpha -converting-enzyme (TACE or 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). TACE/ADAM17 cleaves the 26 kDa form at the Ala76-Val77 bond to produce the 17 kDa form (6, 16). ADAM10 processes the 26 kDa form at the same site as TACE (17). ADAM9 cleaves the 26 kDa form at alternative sites, Ala74-Glu75 and Ser79-Ser80 (18). The use of the recombinant Pro-TNF-alpha fusion protein as a protein substrate for Pro-TNF-alpha processing proteases has been tested with recombinant TACE/ADAM17 (R&D Systems, Catalog # 930-ADB), ADAM10 (Catalog # 936-AD and 946-AD), or ADAM9 (Catalog # 949-AD). The disappearance of the fusion protein (45 kDa) and the appearance of the mature TNF-alpha (17 kDa) were followed by Western blot analysis using an anti-human TNF-alpha polyclonal antibody (Catalog # AF210 or BAF210).

 

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. Marcia, M.L. et al. (1997) Nature 385:733.
17. Rosendahl, M.S. et al. (1997) J. Biol. Chem. 272:24588.
18. Roghani, M. et al. (1999) J. Biol. Chem. 274:3531.

Publications for TNF-alpha (1012-PS)(1)

Publication using 1012-PS

• JB Schaal, DQ Tran, A Subramania, R Patel, T Laragione, KD Roberts, K Trinh, P Tongaonkar, PA Tran, D Minond, GB Fields, P Beringer, AJ Ouellette, PS Gulko, ME Selsted Suppression and resolution of autoimmune arthritis by rhesus ?-defensin-1, an immunomodulatory macrocyclic peptide PLoS ONE, 2017-11-16;12(11):e0187868. 2017-11-16 [PMID: 29145473] (Bioassay)

FAQs for TNF-alpha (1012-PS). (Showing 1 - 4 of 4 FAQ).

1. I need to know about TNF-alpha antibody kits for mice.
   • 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.
   • 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?
   • 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?
   • 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()