Rat MIP-2 ELISA Assay
The Rat MIP-2 ELISA Assay is For Research Use Only
Size: 1×96 wells
Sensitivity: 15 pg/mL
Dynamic Range: 62.5 – 2000 pg/ml
Incubation Time: 3.5 hours
Sample Type: Serum, Plasma, Cell Culture
Sample Size: 100 µl
Alternative Names: Macrophage Inflammatory Protein
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.
Macrophage inflammatory protein 2 (MIP-2) was originally identified as a heparin binding protein secreted from a murine macrophage cell line in response to endotoxin stimulation. The protein is produced by a variety of cell types, including intestinal epithelium, macrophages, astrocytes and fibroblasts. Homologs of the mouse MIP-2 have been identified in human, rat and cotton rat. MIP-2 is an ELR-containing member of the alpha (CXC) subfamily of chemokines. The cotton rat MIP-2 gene encodes a 101 amino acid (aa) residue precursor protein with a 28 aa putative signal peptide that is cleaved to generate the 73 aa mature protein. Mature cotton rat MIP-2 has two intrachain disulfide bonds and no potential glycosylation sites. It shares approximately 79% and 85% aa sequence identity with rat CINC-3 and mouse MIP-2, respectively. MIP-2 is a potent neutrophil attractant and activator. MIP-2 binds and activates the chemokine receptor CXCR-2. The recombinant cotton rat MIP-2 has been shown to bind and activate the human CXCR-2.
Mouse MIP-2 ELISA Assay
Rat TNF-Alpha ELISA Assay
The Eagle Biosciences Rat Macrophage inflammatory protein-2 (MIP-2) ELISA Assay Kit employs the quantitative sandwich enzyme immunoassay technique. A monoclonal antibody specific for MIP-2 has been pre-coated onto a microplate. Standards and samples are pipetted into the wells and any MIP-2 present is bound by the immobilized antibody. Following incubation unbound samples are removed during a wash step, and then a detection antibody specific for MIP-2 is added to the wells and binds to the combination of capture antibody-MIP-2 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 MIP-2 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 MIP-2 standard dilutions and MIP-2 sample concentration determined.
- 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 37C.
- Aspirate each well and wash, repeating the process three times for a total of four washes. Wash by filling each well with Wash Buffer (350 µl) 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 37C.
- 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 37C 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 37C. 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)
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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