Recombinant Human TNF‑α

Catalog Number: AT-0001H-1000/CF

DESCRIPTION

Human tumor necrosis factor alpha (TNF-α), also known as cachectin and TNFSF2, is the prototypic ligand of the TNF superfamily. TNF-α is produced by a wide variety of immune, epithelial, endothelial, and tumor cells^1,2. It is a primary mediator of numerous immunologic functions, including hemorrhagic tumor necrosis/cytotoxicity, inflammation and regulation of antiviral and immune proliferative and activation responses. As a central player in the cytokine network, TNF-α has been implicated in a variety of disease states, including cachexia, endotoxic (septic) shock, acute respiratory distress syndrome and a number of necrotic, proliferative and autoimmune diseases. Human TNF-α encoded by a 7124 gene, is a type II integral membrane protein consisiting of a 35 amino acid (aa) cytoplasmic domain, a 21 aa transmembrane segment, and a 177 aa extracellular domain (ECD). Within the ECD, human TNF-α shares 97% aa sequence identity with rhesus and 71%-92% with bovine, canine, cotton rat, equine, feline, mouse, porcine, and rat TNF-α. TNF-α is assembled intracellularly to form a noncovalently linked homotrimer which is expressed on the cell surface^3,4. Shedding of membrane bound TNF-α by TACE/ADAM17 releases the bioactive cytokine, a 60 kDa soluble trimer of the TNF-α extracellular domain^5-7. TNF-α binds the ubiquitous 55-60 kDa TNFR I^8,9 and the hematopoietic cell-restricted 80 kDa TNFR II, both of which are also expressed as homotrimers^1,2,10. The TNF-α transmembrane protein is proteolytically cleaved to yield a soluble protein, which subsequently forms a non-covalently linked homotrimer in solution¹¹. The individual subunits of this homotrimer have a relative molecular mass each of 20 KDa. TNF-α binds two receptors TNFR I and TNFR II inducing NF-κB and MAPK signaling pathways. Recombinant human TNF-α is intended for use in cell culture applications.

SPECIFICTION

Recombinant human TNF-α is produced in Chinese hamster ovary (CHO) cells transfected with the ECD sequence of the human TNF-α gene with an 8-His tag at C-terminals.

Source: Mammalian; Chinese hamster ovary (CHO) cells

Species: Human; Gene ID: 7124; UniProt ID: P01375   

Molecular weight: 20.6 kDa including tags

Tags: 8-His tag at C-terminals

Concentration: determined by A₂₈₀ with the Extinction coefficient of 1.044 l/g∙ cm^-1 to produce 1 mg/vial, 200 μg/vial product form.

Amino acids: encoding the extracellular (57-233) domain of human TNF with an 8-His tag at C-terminals.

Amino Acid Sequence:

GPQREEFPRDLSLISPLAQAVRSSSRTPSDKPVAHVVANPQAEGQLQWLN

RRANALLANGVELRDNQLVVPSEGLYLIYSQVLFKGQGCPSTHVLLTHTIS

RIAVSYQTKVNLLSAIKSPCQRETPEGAEAKPWYEPIYLGGVFQLEKGDRL

SAEINRPDYLDFAESGQVYFGIIALHHHHHHHH

Formulation: Carrier free recombinant human TNF-α was lyophilized from a 0.22 mm filtered solution in sterile ammonium bicarbonate buffer solution (pH 7.4) (Catalog # AT-0001H/CF; carrier free product formulation) or Recombinant human TNF-α was lyophilized from a 0.22 mm filtered solution in sterile ammonium bicarbonate buffer solution (pH 7.4) and 5% trehalose containing (Catalog # AT-0001H; Stabilier product formulation).

Quality control:

–  Purity greater than 98% as determined by SDS-PAGE.

– Homotrimer conformation as determined by SEC chromatography.

–  TNF-α ELISA for TNF-α folding validation. -Biological activity is tested using L929 cells for cytotoxicity assay. -Endotoxin levels <0.05 EU/μg as determined using a kinetic chromogenic LAL assay.

DATA

This protocol uses Cell Proliferation Assay to determine bioactivity of TNF-α.

1. Maintain and culture L929 cells to confluence in RPMI 1640 complete medium (medium contain 10%  heat inactivated FBS).
2. Trypsinization L929 cell and plating cell number 2x 10⁴/well  in 96-well microplate.
3. Incubate for 16–20 hours (37°C in a 5% CO2).
4. Prepare serial dilutions of TNF-α (0.01 pg/mL to 9 ng/mL) in fresh RPMI complete medium containing 1μg/mL actinomycin D.
5. Remove original medium and add 100 μL/well of these medium with TNF-α dilutions.
6. Incubate the plate for 24 hours at 37°C in a 5% CO2, humidified atmosphere.
7. Remove the medium and add 100 μL/well of fresh medium with cell proliferation reagent WST-1 (Roche), each well contain 90 μL medium and 10 μL WST-1.
8. Incubate the plate for 4 hour at 37°C in a 5% CO2, humidified atmosphere.
9. Shake thoroughly for 1 min on a shaker. 10.Record the absorbance at 450nm using an ELISA plate reader.

PREPARATION AND STORAGE

Recombinant human TNF-α in lyophilized carrier free powder

  form is shipped at room temperature.
– It should be stored at -20 °C; stored at -80^oC upon receiving for

  long term storage.

– Preparation of stock solution (100 μg/ml):
   1. Add 10 ml endotoxin-free water to 1000 μg of
       recombinant human TNF-α.
   2. Mix by pipetting. Do not vortex.
   3. Prepare aliquots of recombinant human TNF-α. Avoid
       freeze-thaw cycles.
   4. Further dilutions can be prepared in the appropriate
– Note: Avoid repeated freeze-thaw cycles.

REFERENCES

1.Zelova H, Hosek J. 2013. Inflamm Res 62: 641-51.
2.Juhasz K, Buzas K, Duda E. 2013. Expert Rev Clin Immunol 9: 335-48.
3.Tang P, Hung MC, Klostergaard J. 1996. Biochemistry 35: 8216-25.
4.Perez C, Albert I, DeFay K, Zachariades N, Gooding L, Kriegler M. 1990. Cell 63: 251-8.
5.Black RA, Rauch CT, Kozlosky CJ, Peschon JJ, Slack JL, et al. 1997. Nature 385: 729-33.
6.Moss ML, Jin SL, Milla ME, Bickett DM, Burkhart W, et al. 1997. Nature 385: 733-6.
7.Gearing AJ, Beckett P, Christodoulou M, Churchill M, Clements J, et al. 1994. Nature 370: 555-7.
8.Schall TJ, Lewis M, Koller KJ, Lee A, Rice GC, et al. 1990. Cell 61: 361-70.
9.Loetscher H, Pan YC, Lahm HW, Gentz R, Brockhaus M, et al. 1990. Cell 61: 351-9.
10.Loetscher H, Gentz R, Zulauf M, Lustig A, Tabuchi H, et al. 1991. J Biol Chem 266: 18324-9.
11.Kriegler M, Perez C, DeFay K, Albert I, Lu SD. 1988. Cell 53: 45-53.

RELATED PRODUCTS

Product	Catalog Code
Anti-hTNF-α-hIgG1	AT-hTNFα-Ab001
Human TNF-α ELISA Set	AT-hTNFα-ELISA001
Recombinant Human TNF‑α	AT-0001H-200/CF
Recombinant Human TNF‑α	AT-0001H-50
Recombinant Human TNF‑α	AT-0001H-200
Recombinant Human TNF‑α	AT-0001H-1000

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