Rat TNF-Alpha ELISA Assay
The Rat TNF-Alpha ELISA Assay is For Research Use Only
Size: 1×96 wells
Sensitivity: 15 pg/mL
Dynamic Range: 31.25 – 2000 pg/ml
Incubation Time: 3.5 hours
Sample Type: Serum, Plasma, Cell Culture
Sample Size: 100 µl
Alternative Names: Rodent TNF-Alpha, TNF-α, Tumor Necrosis Factor alpha, TNFa
SAMPLE COLLECTION AND STORAGE
1. Cell Culture Supernates – Remove particulates by centrifugation.
2. Serum – Use a serum separator tube (SST) and allow samples to clot for 30 minutes before centrifugation for 15 minutes at approximately 1000 x g. Remove serum, avoid hemolysis and high blood lipid samples.
3. Plasma – Recommended EDTA as an anticoagulant in plasma. Centrifuge for 15 minutes at 1000 x g within 30 minutes of collection.
4. Assay immediately or aliquot and store samples at -20°C. Avoid repeated freeze-thaw cycles.
5. Dilute samples at the appropriate multiple (recommended to do pre-test to determine the dilution factor).
Note: Normal rat serum or plasma samples are suggested to make a 1:2 dilution.
The Rat TNF Alpha (TNF-α) ELISA Assay Kit employs the quantitative sandwich enzyme immunoassay technique. A monoclonal antibody specific for TNF Alpha has been pre-coated onto a microplate. Standards and samples are pipetted into the wells and any TNF Alpha present is bound by the immobilized antibody. Following incubation unbound samples are removed during a wash step, and then a detection antibody specific for TNF Alpha is added to the wells and binds to the combination of capture antibody TNF-alpha in sample. Following a wash to remove any unbound combination, and enzyme conjugate is added to the wells. Following incubation and wash steps a substrate is added. A colored product is formed in proportion to the amount of TNF Alpha present in the sample. The reaction is terminated by addition of acid and absorbance is measured at 450nm. A standard curve is prepared from seven TNF Alpha (TNF-α) standard dilutions and TNF Alpha sample concentration determined.
TNF-Alpha ELISA Assay Kit
Mouse TNF-Alpha ELISA Assay
- Prepare all reagents and working standards as directed in the previous sections.
- Determine the number of microwell strips required to test the desired number of samples plus appropriate number of wells needed for running blanks and standards. Remove extra microwell strips from holder and store in foil bag with the desiccant provided at 2-8°C sealed tightly.
- Add 100 µL of Standard, control, or sample, per well. Cover with the adhesive strip provided. Incubate for 1.5 hours at 37°C.
- Aspirate each well and wash, repeating the process three times for a total of four washes. Wash by filling each well with Wash Buffer (350µL) using a squirt bottle, manifold dispenser or auto-washer. Complete removal of liquid at each step is essential to good performance. After the last wash, remove any remaining Wash Buffer by aspirating or decanting. Invert the plate and blot it against clean paper towels.
- Add 100 µL of the working solution of Biotin-Conjugate to each well. Cover with a new adhesive strip and incubate 1 hour at 37°C.
- Repeat the aspiration/wash.
- Add 100 µL of the working solution of Streptavidin-HRP to each well. Cover with a new adhesive strip and incubate for 30 minutes at 37°C Avoid placing the plate in direct light.
- Repeat the aspiration/wash.
- Add 100 µL of Substrate Solution to each well. Incubate for 10-20 minutes at 37°C. Avoid placing the plate in direct light.
- Add 100 µL of Stop Solution to each well. Gently tap the plate to ensure thorough mixing.
- Determine the optical density of each well immediately, using a microplate reader set to 450 nm.(optionally 630nm as the reference wave length; 610-650nm is acceptable)
TNF Alpha (TNF-α) is a pleiotropic cytokine that plays a central role in inflammation and apoptosis (1-4). TNF-α is produced by activated macrophages and other cell types including T and B cells, NK cells, LAK cells, astrocytes, endothelial cells, smooth muscle cells and some tumor cells (5-8). Rat TNF-α cDNA encodes a 235 amino acid (aa) residue type II membrane protein (9). The 156 aa residue soluble TNF-α is released from the C-terminus of the membrane-anchored TNF-α by TNF-α-converting enzyme (TACE), a matrix metalloprotease (10).
The membrane-anchored form of TNF-α has been shown to have lytic activity and may also have an important role in intercellular communication (11). The biologically active TNF-α has been shown to exist as a trimer (12, 13).TNF-α is reported to promote inflammatory cell infiltration by upregulating leukocyte adhesion molecules on endothelial cells, serve as a chemotactic agent for monocytes, and activate phagocyte killing mechanisms (14). Deficiencies in either TNF-α or its receptors can increase susceptibility to infection by intracellular pathogens (15-16). TNF-α may also play a role in lymphoid tissue development. Knockout mice lack splenic B cell follicles and the ability to form germinal centers (17, 18). Other potential physiological roles for TNF-α and its receptors include regulating the differentiation of hematopoietic stem and progenitor cells (19-21).
Please note: All documents above are for reference use only and should not be used in place of the documents included with this physical product. If digital copies are needed, please contact us.
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