Recombinant human TNF-α protein - Bioactive cytokine

Human TNF-α protein - Mammalian cell-expressed, tag-free, with HSA

Recombinant human TNF-α is a high-quality and biologically active cytokine, validated using proprietary TNF-α reporter cells. This pro-inflammatory cytokine of the TNF superfamily is produced in CHO cells to ensure protein glycosylation and bona fide 3D structure.

Recombinant human TNF-α can be used together with HEK-Blue™ TNF-α cells for the screening of inhibitory molecules, such as Adalimumab, a therapeutic monoclonal antibody that targets TNF-α and prevents its binding to its receptor (see figures).

Key features

• Each lot is validated using HEK-Blue™ TNF-α cells 
• Endotoxin ≤ 0.1 EU/µg
• 0.2 µm sterile-filtered

Applications

• TNF-α detection and quantification assays (positive control)
• Screening and release assays for antibodies blocking TNF-α signaling
• Screening and release assays for engineered TNF-α proteins

Tumor necrosis factor-alpha (TNF-α) is a pleiotropic cytokine that plays a central role in inflammation, cell apoptosis, and immune system development. This potent mediator of inflammation is implicated in the pathogenesis of several autoimmune and inflammatory diseases such as rheumatoid arthritis and inflammatory bowel disease.

 More details

InvivoGen’s products are for internal research use only, and not for human or veterinary use.

Figures

SDS-PAGE of the recombinant human (h)TNF-a protein. 1 μg of hTNF-α was loaded on a 12% Mini-PROTEAN® TGX Stain-Free™ Precast Gel (Bio-Rad). Detection was performed as per the manufacturer’s instructions. A band was detected at ~17 kDa.

Dose-response of HEK-Blue™ TNF-α cells to recombinant TNF-α. Cells were stimulated with increasing concentrations of recombinant human TNF-α. After overnight incubation, the NF-κB/AP1-induced SEAP activity was determined using QUANTI-Blue™, a SEAP detection reagent. Data are shown as optical density (OD) at 650 nm (mean ± SEM).

Dose-dependent inhibition of HEK-Blue™ TNF-α cell response using Adalimumab biosimilar. Increasing concentrations of Anti-hTNF-α Adalimumab biosimilar (1 ng/ml - 1 µg/ml) were incubated with recombinant human TNF-α (30 ng/ml) for 30 minutes before the addition of HEK-Blue™ TNF-α cells. After overnight incubation, SEAP activity in the cell culture supernatant was assessed using QUANTI-Blue™ Solution. Data are shown in percentage of activity (mean ± SEM).

Specifications

Source: Chinese hamster ovary (CHO) cells

Species: Human

Alternative name: Cachectin, Tumor necrosis factor ligand superfamily member 2

Carrier: HSA (human serum albumin)

Tag: Tag-free

Accession number: P01375

Molecular weight: ~ 17 kDa (SDS-PAGE)

Solubility: 100 μg/ml in water

Formulation: Phosphate buffer saline (pH 7.4), 5% saccharose, 0.1% HSA

Sterility: 0.2 µm filtration

Form:  Lyophilized

Reconstitution buffer:  Endotoxin-free water (provided)

Purity:  ≥97% (SDS-PAGE)

Endotoxin: The absence of bacterial contamination (e.g. lipoproteins and endotoxins) has been confirmed using HEK-Blue™ TLR2 and HEK‑Blue™ TLR4 cells.

Tested applications:  Cellular assays

Quality control: Each lot is functionally tested and validated

Contents

Recombinant human TNF-α is provided as a lyophilized powder and is available in two quantities:

• rcyc-htnfa: 20 µg
• rcyc-htnfa: 100 µg (5 x 20 µg)
• 1.5 ml endotoxin-free water for rcyc-htnfa and rcyc-htnfa-5

Recombinant human TNF-α is shipped at room temperature.

Upon receipt, the product should be stored at -20°C.

 Avoid repeated freeze-thaw cycles.

Details

Tumor necrosis factor-alpha (TNF-α) is a pleiotropic cytokine involved in necrotic and apoptotic cell death, cellular differentiation, inflammation, and regulation of immune cell activity [1]. Notably, deregulated TNF-α production has been implicated in a variety of conditions, including autoimmune and inflammatory diseases [1].

TNF-α is mainly produced by activated monocytes, macrophages, and T cells. It is first synthesized as a membrane-bound molecule that forms a compact homotrimer through non-covalent interactions. The trimeric membrane-bound form is cleaved by tumor necrosis factor-alpha converting enzyme (TACE) releasing the soluble trimer [2]. Both the membrane-bound and soluble TNF-α bind homotrimeric transmembrane receptors, TNFR1 or TNFR2, triggering signaling pathways that involve TRADD, TRAF2, and RIP, and leading to the activation of NF-κB and MAPK pathways.

1. Steeland S. et al., 2018. A new venue of TNF targeting. Int. J. Mol. Sci. 19:1442.
2. Brenner D. et al., 2015. Regulation of tumour necrosis factor signalling: live or let die. Nat Rev Immunol. 15(6):362-74.