Recombinant Human TNF-alpha Pro form Protein

Product Discontinued

Recombinant Human TNF-alpha Pro form Protein Summary

• Description: Recombinant Human Pro TNF-a Bacterial Protein Fusion Partner protein
  
  Source: E. coli derived
  
  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-a to produce mature TNF-a.
  
  Assay Procedure
  
  Materials:
  
  • Assay Buffer: 50 mM Tris, pH 8.0
  • Recombinant Human Pro TNF-a Fusion Protein (rhPro-TNF-a) (Catalog # 1012-PS)
  • Recombinant Human TACE/ADAM17 (rhTACE) (Catalog # 930-ADB)
  • Positive Control: Recombinant Human TNF-a (rhTNF-a) (Catalog # 210-TA)
  • Goat Anti-Human TNF-a Biotinylated Antigen Affinity-purified Polyclonal Antibody (Catalog # BAF210)
  • SDS-PAGE followed by Western Blotting
  1. Dilute rhPro-TNF-a to 0.2 mg/mL in Assay Buffer.
  2. Dilute rhTACE to 0.2 mg/mL in Assay Buffer.
  3. Prepare the following vials for a final volume of 20 uL.
  1. 10 uL of rhPro-TNF-a at 0.2 mg/mL + 10 uL of rhTACE at 0.2 mg/mL
  2. 10 uL of rhPro-TNF-a at 0.2 mg/mL + 10 uL of Assay Buffer (control-with incubation)
  3. 10 uL of rhPro-TNF-a at 0.2 mg/mL + 10 uL of Assay Buffer (control-without incubation)
     Note: At this point the concentration of rhPro-TNF-a in the reaction tubes is 0.1 mg/mL.
  4. Incubate vials at 37C overnight (except for the non-incubated control, store at =-20C).
  5. Stop the reactions by adding the reducing gel loading buffer for SDS-PAGE to all vials. Heat the samples at 100C for 3-5 minutes.
  1. Dilute the reaction tubes (and control without incubation) to 50 ng/15 uL in reducing sample buffer.
  6. Prepare a sample of Positive Control at 5 ng/15 uL in reducing sample buffer. Heat the sample at 100C for 3-5 minutes.
  7. Load the samples on a 15% gel.
  1. 50 ng/lane (15 uL) of rhPro-TNF-a of the incubated reactions (including the control with incubation).
  2. 50 ng/lane (15 uL) of rhPro-TNF-a of the control (without incubation).
  3. 5 ng/lane (15 uL) of Positive Control (Catalog # 210-TA).
  8. Follow SDS-PAGE/Western Blotting procedures.
  1. Use the Biotinylated Anti-human TNF-a/TNFSF1A antibody at 0.1 ug/mL. (Catalog # AF-210-NA may be used in place of # BAF210.)
  9. Visually determine processing of rhPro-TNF-a to the mature form by rhTACE by comparing the incubated reactions to the Positive Control.
• Source: E. coli
• Protein/Peptide Type: Partial Recombinant Protein
• Gene: TNF
• Purity: >90%, by SDS-PAGE under reducing conditions and visualized by silver stain.
• Endotoxin Note: <1.0 EU per 1 ug of the protein by the LAL method.

Applications/Dilutions

• 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.

Packaging, Storage & Formulations

• Storage: Protect from light. Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
  • 12 months from date of receipt, -20 to -70 °C as supplied.
  • 3 months, -20 to -70 °C under sterile conditions after opening.
• Buffer: 0.2 um 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 200 ug/mL in sterile, deionized water.

Alternate Names for Recombinant Human TNF-alpha Pro form Protein

• APC1 protein
• Cachectin
• Cachetin
• DIF
• TNF
• TNF, monocyte-derived
• TNFA
• TNF-A
• TNFalpha
• TNF-alpha
• TNF-alpha/TNFSF2
• 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 (TNF)-alpha is a pro-inflammatory cytokine belonging to the TNF superfamily that is secreted by monocytes/macrophages, T cell, and natural killer (NK), among others (1). TNF-alpha is synthesized as a 233 amino acid (aa) transmembrane protein (mTNF-alpha) with a theoretical molecular weight (MW) of 26 kDa (1,2) that forms a homotrimer. mTNF-alpha is cleaved by TNF-alpha converting enzyme (TACE) and released in its 157 aa, 17 kDa soluble form (sTNF-alpha) (1-5). Both mTNF-alpha and sTNF-alpha are capable of binding type 1 TNF receptors (TNFR1), whereas mTNF-alpha predominately binds to TNFR2 (1,2). TNF-alpha binding to its receptors causes receptor recruitment of adaptor proteins, formation of signaling complexes, and downstream signaling cascades (e.g. MAPK, NF-kappaB, and Caspase-8), leading to distinct cellular responses such as survival, proliferation, inflammation, necroptosis, and apoptosis (1-5).

TNF-alpha is critical for normal immune response; however, dysregulation of TNF-alpha production can result in various pathologies (2,4,5). Excessive production of pro-inflammatory cytokines including interleukin 1 (IL-1), IL-6, and TNF-alpha has been implicated in an array of autoimmune diseases like rheumatoid arthritis (RA), inflammatory bowel disease (IBD), and psoriasis (2,4,5). Anti-TNF monoclonal antibodies, including Infliximab, and soluble TNFR have been approved for the treatment of autoimmune and TNF-mediated diseases (5). Additionally, data suggests that TNF inhibitors can be beneficial for treating patients experiencing immune-related adverse events associated with immune checkpoint inhibitor cancer treatment (6).

References

1. Holbrook J, Lara-Reyna S, Jarosz-Griffiths H, McDermott M. Tumour necrosis factor signalling in health and disease. F1000Res. 2019;8:F1000 Faculty Rev-111. https://doi.org/10.12688/f1000research.17023.1

2. Jang DI, Lee AH, Shin HY, et al. The Role of Tumor Necrosis Factor Alpha (TNF-alpha) in Autoimmune Disease and Current TNF-alpha Inhibitors in Therapeutics. Int J Mol Sci. 2021;22(5):2719. https://doi.org/10.3390/ijms22052719

3. Horiuchi T, Mitoma H, Harashima S, Tsukamoto H, Shimoda T. Transmembrane TNF-alpha: structure, function and interaction with anti-TNF agents. Rheumatology (Oxford). 2010;49(7):1215-1228. https://doi.org/10.1093/rheumatology/keq031

4. Webster JD, Vucic D. The Balance of TNF Mediated Pathways Regulates Inflammatory Cell Death Signaling in Healthy and Diseased Tissues. Front Cell Dev Biol. 2020;8:365. https://doi.org/10.3389/fcell.2020.00365

5. Kalliolias GD, Ivashkiv LB. TNF biology, pathogenic mechanisms and emerging therapeutic strategies. Nat Rev Rheumatol. 2016; 12(1):49-62. https://doi.org/10.1038/nrrheum.2015.169

6. Chen AY, Wolchok JD, Bass AR. TNF in the era of immune checkpoint inhibitors: friend or foe?. Nat Rev Rheumatol. 2021;17(4):213-223. doi:10.1038/s41584-021-00584-4

Limitations

This product is for research use only and is not approved for use in humans or in clinical diagnosis. Peptides and proteins are guaranteed for 3 months from date of receipt.

FAQs for TNF-alpha Partial Recombinant Protein (NB1012-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
• Entrez:
  • PO1375(Human)
  • PO1375(Human)