Cell Signaling Technology

Product Pathways - Growth Factors/Cytokines

Human Tumor Necrosis Factor-α (hTNF-α) #8902

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Source

Recombinant human TNF-α (hTNF-α) Val77-Leu233 (Accession #HUMTNFAB) was produced in E. coli at Cell Signaling Technology.

Molecular Characterization

Recombinant hTNF-α does not have a Met on the amino terminus and has a calculated MW of 17,352. DTT-reduced and non-reduced protein migrate as 18 kDa polypeptides. The expected amino-terminal VRSSS of recombinant hTNF-α was verified by amino acid sequencing. TNF-α is a non-disulfide-linked homotrimer in solution as determined by chemical cross-linking.

Purity

>98% as determined by SDS-PAGE of 6 μg reduced (+) and non-reduced (-) recombinant hTNF-α. All lots are greater than 98% pure.

Bioactivity

The bioactivity of hTNF-α was determined in an L-929 cell viability assay. The ED50 of each lot is between 10-100 pg/ml.

Coomassie Gel

Coomassie Gel

The purity of recombinant hTNF-α was determined by SDS-PAGE of 6 µg reduced (+) and non-reduced (-) recombinant hTNF-α and staining overnight with Coomassie Blue.

Bioactivity

Bioactivity

The viability of L-929 cells treated with increasing amounts of hTNF-α in the presence of 2 ng/ml actinomycin D was determined. Cells were stained with crystal violet at the end of treatment and the OD595 was determined.

Western Blotting

Western Blotting

Western blot analysis of extracts from HeLa cells treated with hTNF-α for 20 minutes, using Phospho-NF-κB p65 (Ser536) (93H1) Rabbit mAb #3033 (upper) and total NF-κB p65 Antibody #3034 (lower).


Endotoxin

Less than 0.01 ng endotoxin/1 μg hTNF-α.

Formulation

With carrier: Lyophilized from a 0.22 μm filtered solution of PBS, pH 7.2 containing 20 μg BSA per 1 μg hTNF-α. Carrier free: Lyophilized from a 0.22 μm filtered solution of PBS, pH 7.2.

Background

TNF-α, the prototypical member of the TNF protein superfamily, is a homotrimeric type-II membrane protein (1,2). Membrane bound TNF-α is cleaved by the metalloprotease TACE/ADAM17 to generate a soluble homotrimer (2). Both membrane and soluble forms of TNF-α are biologically active. TNF-α is produced by a variety of immune cells including T cells, B cells, NK cells and macrophages (1). Cellular response to TNF-α is mediated through interaction with receptors TNF-R1 and TNF-R2 and results in activation of pathways that favor both cell survival and apoptosis depending on the cell type and biological context. Activation of kinase pathways (including JNK, ERK (p44/42), p38 MAPK and NF-κB) promotes the survival of cells, while TNF-α mediated activation of caspase-8 leads to programmed cell death (1,2). TNF-α plays a key regulatory role in inflammation and host defense against bacterial infection, notably Mycobacterium tuberculosis (3). The role of TNF-α in autoimmunity is underscored by blocking TNF-α action to treat rheumatoid arthritis and Crohn’s disease (1,2,4).

  1. Aggarwal, B.B. (2003) Nat Rev Immunol 3, 745-56.
  2. Hehlgans, T. and Pfeffer, K. (2005) Immunology 115, 1-20.
  3. Lin, P.L. et al. (2007) J Investig Dermatol Symp Proc 12, 22-5.
  4. Brennan, F.M. and McInnes, I.B. (2008) J Clin Invest 118, 3537-45.

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