Recombinant Pro-TNF-alpha Fusion Protein, CF

R&D Systems | Catalog # 1012-PS

R&D Systems
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Key Product Details

  • R&D Systems E. coli-derived Recombinant Pro-TNF-alpha Fusion Protein (1012-PS)
  • Quality control testing to verify active proteins with lot specific assays by in-house scientists
  • All R&D Systems proteins are covered with a 100% guarantee

Source

E. coli

Accession Number

P01375

Applications

Enzyme Activity
View all TNF-alpha Proteins and Enzymes »

Product Specifications

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

Purity

>90%, by SDS-PAGE under reducing conditions and visualized by silver stain.

Endotoxin Level

<1.0 EU per 1 μg of the protein by the LAL method.

N-terminal Sequence Analysis

Bacterial Protein Fusion Partner

Predicted Molecular Mass

45 kDa

SDS-PAGE

42 kDa, reducing conditions

Activity

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. 

Formulation, Preparation, and Storage

1012-PS
Formulation Lyophilized from a 0.2 μm filtered solution in Urea, NaCl, NaH2PO4 and DTT.
Reconstitution

Reconstitute at 0.2 mg/mL in sterile, deionized water.
Shipping The product is shipped with polar packs. Upon receipt, store it immediately at the temperature recommended below.
Stability & 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.

Calculators

The reconstitution calculator allows you to quickly calculate the volume of a reagent to reconstitute your vial. Simply enter the mass of reagent and the target concentration and the calculator will determine the rest.

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Background: TNF-alpha

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).

 

References

  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.

Long Name

Tumor Necrosis Factor alpha

Alternate Names

Cachetin, DIF, TNF, TNF-A, TNFA, TNFalpha, TNFG1F, TNFSF1A, TNFSF2

Entrez Gene IDs

7124 (Human); 21926 (Mouse); 24835 (Rat); 397086 (Porcine); 280943 (Bovine); 403922 (Canine); 102139631 (Cynomolgus Monkey); 100033834 (Equine); 493755 (Feline); 100009088 (Rabbit)

Gene Symbol

TNF

UniProt

P01375

Additional TNF-alpha Products

Product Documents for Recombinant Pro-TNF-alpha Fusion Protein, CF

Certificate of Analysis

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Product Specific Notices for Recombinant Pro-TNF-alpha Fusion Protein, CF

For research use only

Citations for Recombinant Pro-TNF-alpha Fusion Protein, CF

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Protocols

View specific protocols for Recombinant Pro-TNF-alpha Fusion Protein, CF (1012-PS):

Materials
  • 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.

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