Recombinant Human TNF-alpha GMP Protein, CF

Animal-Free.
  
  • Purity
    >97%, by SDS-PAGE with silver staining.
  • Endotoxin Level
    <0.10 EU per 1 μg of the protein by the LAL method.
  • Activity
    Measured in a cytotoxicity assay using L‑929 mouse fibroblast cells in the presence of the metabolic inhibitor actinomycin D. Matthews, N. and M.L. Neale (1987) in Lymphokines and Interferons, A Practical Approach. Clemens, M.J. et al. (eds): IRL Press. 221. The ED50 for this effect is 25-100 pg/mL.
    The specific activity of recombinant human TNF-alpha GMP is approximately 7.6 x 104 IU/μg, which is calibrated against human TNF-alpha WHO International Standard (NIBSC code: 88/786).
  • Source
    E. coli-derived Val77-Leu233, with and without an N-terminal Met Produced using non-animal reagents in an animal-free laboratory.
    Manufactured and tested under cGMP guidelines.
  • Accession #
  • N-terminal Sequence
    Analysis
    Met-Val77-Arg-Ser-Ser-Ser-Arg-Thr-Pro-Ser
    Val77-Arg-Ser-Ser-Ser-Arg-Thr-Pro-Ser-Asp
  • Predicted Molecular Mass
    17 kDa
  • SDS-PAGE
    17 kDa, reducing conditions
210-GMP
 
Formulation Lyophilized from a 0.2 μm filtered solution in PBS.
Reconstitution Reconstitute at 100-200 μg/mL in PBS.
Shipping The product is shipped at ambient temperature. Upon receipt, store it immediately at the temperature recommended below.
Stability & Storage: Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
  • A minimum of 6 months when stored at ≤ -20 °C as supplied. Refer to lot specific COA for the Use by Date.
  • 1 month, 2 to 8 °C under sterile conditions after reconstitution.
  • 3 months, ≤ -20 °C under sterile conditions after reconstitution.
Data Images

GMP-grade Recombinant Human TNF-alpha (Catalog # 210-GMP) induces cytotoxicity in the L-929 mouse fibroblast cell line in the presence of the metabolic inhibitor actinomycin D. The ED50 is
25-100 pg/mL.

1 μg/lane of GMP-grade Recombinant Human TNF-alpha (Catalog # 210-GMP) was resolved by SDS-PAGE with silver staining, under reducing (R) conditions, showing a single band at 17 kDa.

Mass Spectrometry
MALDI-TOF analysis of GMP-grade Recombinant Human TNF-alpha (Catalog # 210‑GMP). The major peak at 17348 corresponds to the calculated molecular mass without an N-terminal Met, 17353 Da. The minor peak at 17480 corresponds to the calculated molecular mass with an N-terminal Met, 17484 Da. The peak at 17565 is a matrix-associated artifact of the MALDI-TOF.
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 TACE/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).
  • 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.
  • Long Name:
    Tumor Necrosis Factor alpha
  • Entrez Gene IDs:
    7124 (Human); 21926 (Mouse); 24835 (Rat); 397086 (Porcine); 280943 (Bovine); 403922 (Canine); 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; 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

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