Equine TNF-alpha Antibody

Catalog #: AF1814 Datasheet

Catalog #	Availability	Size / Price	Qty
AF1814
AF1814-SP

Cytotoxicity Induced by TNF‑ alpha and Neutralization by Equine TNF‑ alpha Antibody.

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Equine TNF-alpha Antibody Summary

Species Reactivity

Equine

Specificity

Detects equine TNF‑alpha in direct ELISAs and Western blots. In Western blots, approximately 35% cross-reactivity with recombinant mouse TNF-alpha and recombinant rat TNF-alpha is observed.

Source

Polyclonal Goat IgG

Purification

Antigen Affinity-purified

Immunogen

E. coli-derived recombinant equine TNF-alpha
Leu78-Leu234
Accession # NP_001075288

Formulation

Lyophilized from a 0.2 μm filtered solution in PBS with Trehalose. *Small pack size (SP) is supplied either lyophilized or as a 0.2 µm filtered solution in PBS.

Endotoxin Level

<0.10 EU per 1 μg of the antibody by the LAL method.

Label

Unconjugated

Applications

Recommended Concentration

Sample

Western Blot

0.1 µg/mL

Recombinant Equine TNF‑ alpha (Catalog # 1814-ET)

Immunocytochemistry

5-15 µg/mL

See below

Neutralization

Measured by its ability to neutralize TNF‑ alpha -induced cytotoxicity in the L‑929 mouse fibroblast cell line. The Neutralization Dose (ND₅₀) is typically 0.01-0.03 µg/mL in the presence of 1 ng/mL Recombinant Equine TNF‑ alpha and 1 µg/mL actinomycin D.

Please Note: Optimal dilutions should be determined by each laboratory for each application. General Protocols are available in the Technical Information section on our website.

Scientific Data

Neutralization

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Cytotoxicity Induced by TNF‑ alpha and Neutralization by Equine TNF‑ alpha Antibody. Recombinant Equine TNF-a (Catalog # 1814-ET) induces cytotoxicity in the the L-929 mouse fibroblast cell line in a dose-dependent manner (orange line), as measured by Resazurin (Catalog # AR002). Cytotoxicity elicited by Recombinant Equine TNF-a (1 ng/mL) is neutralized (green line) by increasing concentrations of Goat Anti-Equine TNF-a Antigen Affinity-purified Polyclonal Antibody (Catalog # AF1814). The ND₅₀ is typically 0.01-0.03 µg/mL in the presence of the metabolic inhibitor actinomycin D (1 µg/mL).

Immunocytochemistry

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TNF‑ alpha in Equine PBMCs. TNF-a was detected in immersion fixed equine peripheral blood mononuclear cells (PBMCs) using Goat Anti-Equine TNF-a Antigen Affinity-purified Polyclonal Antibody (Catalog # AF1814) at 15 µg/mL for 3 hours at room temperature. Cells were stained using the NorthernLights™ 557-conjugated Anti-Goat IgG Secondary Antibody (red; Catalog # NL001) and counterstained with DAPI (blue). Specific staining was localized to cytoplasm. View our protocol for Fluorescent ICC Staining of Non-adherent Cells.

Preparation and Storage

Reconstitution

Reconstitute at 0.2 mg/mL in sterile PBS.

Shipping

Lyophilized product is shipped at ambient temperature. Liquid small pack size (-SP) is shipped with polar packs. Upon receipt, store immediately at the temperature recommended below.

Stability & Storage

Use a manual defrost freezer and avoid repeated freeze-thaw cycles.

12 months from date of receipt, -20 to -70 °C as supplied.
1 month, 2 to 8 °C under sterile conditions after reconstitution.
6 months, -20 to -70 °C under sterile conditions after reconstitution.

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, apoptosis, and immune system development. TNF-alpha is produced by a wide variety of immune and epithelial cell types (1, 2). Equine TNF-alpha consisits of a 35 amino acid (aa) cytoplasmic domain, a 21 aa transmembrane segment, and a 178 aa extracellular domain (ECD) (3). Within the ECD, equine TNF-alpha shares 69%‑88% aa sequence identity with bovine, canine, cotton rat, feline, human, mouse, porcine, rat, and rhesus TNF-alpha. The 26 kDa type 2 transmembrane protein is assembled intracellularly to form a noncovalently linked homotrimer (4). Ligation of this complex induces reverse signaling that promotes lymphocyte costimulation but diminishes monocyte responsiveness (5). Cleavage of membrane bound TNF-alpha by TACE/ADAM17 releases a 55 kDa soluble trimeric form of TNF-alpha (6, 7). TNF-alpha trimers bind the ubiquitous TNF RI and the hematopoietic cell-restricted TNF RII, both of which are also expressed as homotrimers (1, 8). TNF-alpha regulates lymphoid tissue development through control of apoptosis (2). It also promotes inflammatory responses by inducing the activation of vascular endothelial cells and macrophages (2). TNF-alpha is a key cytokine in the development of several inflammatory disorders (9). It contributes to the development of type 2 diabetes through its effects on insulin resistance and fatty acid metabolism (10, 11).

References

Idriss, H.T. and J.H. Naismith (2000) Microsc. Res. Tech. 50:184.
Hehlgans, T. and K. Pfeffer (2005) Immunology 115:1.
Su, X.Z. et al. (1991) Gene 107:319.
Tang, P. et al. (1996) Biochemistry 35:8216.
Eissner G. et al. (2004) Cytokine Growth Factor Rev. 15:353.
Black, R.A. et al. (1997) Nature 385:729.
Moss, M.L. et al. (1997) Nature 385:733.
Loetscher, H. et al. (1991) J. Biol. Chem. 266:18324.
Clark, I.A. (2007) Cytokine Growth Factor Rev. 18:335.
Romanatto, T. et al. (2007) Peptides 28:1050.
Hector, J. et al. (2007) Horm. Metab. Res. 39:250.

Long Name

Tumor Necrosis Factor alpha

Entrez Gene IDs

7124 (Human); 21926 (Mouse); 24835 (Rat); 397086 (Porcine); 280943 (Bovine); 403922 (Canine); 102139631 (Cynomolgus Monkey); 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; 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