TNF alpha ELISA Kit, Rat

Catalog number: KRC3012

Novex™  Related applications: Protein Assays and Analysis

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Description

The Rat Tumor Necrosis Factor alpha (TNF alpha, TNF) ELISA research-use-only kit is to be used for the quantitative determination of TNF alpha in samples (see sample types indicated) using 96-well plates and a microplate reader. The assay will recognize both natural and recombinant forms of this target.

Performance characteristics

• Sensitivity: <4 pg/mL
• Standard curve range: 11.7‒750 pg/mL
• Sample type(s): serum, plasma, cell culture supernatant
• Specificity: natural and recombinant rat TNF alpha
• Cross-reactivity: see kit manual for cross-species and/or cross-target reactivity
• Sample volume: 100 μL
• Total assay time: 4 hours

Rigorous validation

Each manufactured lot of this ELISA kit is quality tested for criteria such as sensitivity, specificity, precision, and lot-to-lot consistency. See manual for more information on validation.

Principle of the method

The rat TNF alpha solid-phase sandwich ELISA (enzyme-linked immunosorbent assay) is designed to measure the amount of the target bound between a matched antibody pair. A target-specific antibody has been pre-coated in the wells of the supplied microplate. Samples, standards, or controls are then added into these wells and bind to the immobilized (capture) antibody. The sandwich is formed by the addition of the second (detector) antibody, binding to the target on a different epitope from the capture antibody. A conjugated enzyme has been incorporated into the assay. After incubation and washing steps to rid the microplate of unbound substances, a substrate solution is added that reacts with the enzyme-antibody-target complex to produce measurable signal. The intensity of this signal is directly proportional to the concentration of target present in the original specimen.

Target information

Tumor necrosis factor-alpha (TNF-α; Tumor necrosis factor ligand superfamily member 2; cachexin; cachectin; accession number P16599), is a key inflammatory mediator that is implicated in a number of pathologies (including septic shock and autoimmune diseases) as well as normal cell processes (cell growth and homeostasis, immune system function, and angiogenesis). Rat TNF-α bears a high degree of homology with mouse and human TNF-α (94% and 76% for the soluble forms, respectively), and with the other members of the TNF superfamily (LTa, LTb, LIGHT, CD95L, TWEAK, TRAIL, CD40L, CD30L, CD27L, 4-1BBL, Ox40L, TRANCE/RANKL, TL1/VEGI, GITRL, and TALL-1/THANK). As with most members of the TNF superfamily (the exception being the secreted protein LTa), TNF-α is synthesized as a type II transmembrane protein with a receptor binding motif located at the C-terminus. In the rat, the transmembrane form is a 26 kDa protein containing 253 amino acid residues. Cleavage of the membrane-associated form by the metalloprotease TNF-α converting enzyme (TACE, also called ADAM17) releases soluble TNF-α, a 17 kDa protein containing 165 amino acid residues. Both the membrane-associated and the soluble forms are biologically active. While most nucleated cells have the capability to produce TNF-α, major sources of the cytokine include stimulated monocytes and activated lymphocytes. TNF-α production is regulated by 3’ AU-rich elements (ARE) and the transcription factors NFκB, SP-1, AP-2, and IFN-1. Stimuli that enhance TNF-α production include LPS and other pathogen-associated molecular patterns (“PAMPs”), phorbol esters, calcium ionophores, and the cytokines IL-1, IL-2, and GM-CSF. Stimuli that downregulate TNF-α include cyclosporine A, vitamin D3, prostaglandin E2, dexamethasone, and the cytokines IL-6 and TGF-β. Signaling arising from TNF-α results from the binding of trimeric forms of the cytokine to specific receptors, designated TNF-RI (TNF receptor type I, CD120a, p55/60) and TNF-RII (TNF receptor type II, CD120b, p75.80). TNF-RI is expressed by most somatic cell types, while TNF-RII expression appears to be limited to cells of the immune system. TNF-α bound to these receptors imposes trimerization of receptor subunits, inducing receptor cross-talk and activation of intracellular signaling cascades. Intracellular proteins involved in TNF-α signaling include TRADD, RIP, and TRAF2. TNF-α signaling stimulates the activation of MAPK, AP-1, and NFκB pathways. By acting through FADD, TNF-α stimulation activates caspase-8 and apoptosis. Soluble forms of TNF-RI and TNF-RII are present in relatively high levels in serum and plasma. The role of the soluble receptors seems to be in the regulation of the biological activity of TNF-α by buffering the circulating levels. TNF-α, along with the cytokines IL-1 and IL-6, is a key mediator of the acute phase response. In the acute phase response, inflammatory cells such as neutrophils and macrophages at localized sites of injury secrete proinflammatory cytokines into the bloodstream. These cytokines stimulate the production of acute phase proteins by the liver. In the rat, acute phase proteins include fibrinogen, cysteine protease inhibitor, α2 macroglobulin, and α1 acid glycoprotein.

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For Research Use Only. Not for use in diagnostic procedures.

Specifications

Gene Aliases: MGC124630, RATTNF, TNF-alpha, Tnfa
Accession Number: NP_036807.1, NM_012675.3
Shipping Condition: Wet Ice
Marker: TNF-α
Gene ID: 24835
Species: Rat
Gene Symbol: Tnf
Sensitivity: <4 pg⁄mL
Target Gene: tumor necrosis factor
Label or Dye: HRP (Horseradish Peroxidase)
Product Size: 192 assays
Concentration: 15.6-1000 pg⁄ml
Sample Volume: 50 µl
Protein Family: Cytokines & Receptors
Incubation Time: 2.75 hrs
Detection Method: Colorimetric
Research Category: Immunology, Apoptosis
Sample Type (Specific): Plasma, Serum, Supernatants
For Use With (Equipment): Microplate Reader

Contents & storage

Pre-coated plate(s), standard or calibrator, detector antibody, HRP conjugate, diluents, wash buffer, substrate(s), and stop solution. Store kit at 2-8°C. See product manual for detailed contents and storage conditions for maximum stability.

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Manuals & protocols

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