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Recombinant Pro-TNF-alpha Fusion Protein, CF

Catalog #: 1012-PS
1 Citation Datasheet
Catalog # Availability Size / Price Qty
1012-PS-010
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Recombinant Pro-TNF-alpha Fusion Protein, CF Summary

Product Specifications

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.
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. 
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
Analysis
Bacterial Protein Fusion Partner
Predicted Molecular Mass
45 kDa
SDS-PAGE
42 kDa, reducing conditions

Product Datasheets

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1012-PS

Product Datasheet

Carrier Free

What does CF mean?

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.

What formulation is right for me?

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.

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.

Assay Procedure

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.

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

Citation for Recombinant Pro-TNF-alpha Fusion 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.

1 Citation: Showing 1 - 1

  1. Suppression and resolution of autoimmune arthritis by rhesus ?-defensin-1, an immunomodulatory macrocyclic peptide
    Authors: 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
    PLoS ONE, 2017-11-16;12(11):e0187868.
    Applications: Bioassay

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