Human OSMR beta DuoSet ELISA Summary
* Provided that the recommended microplates, buffers, diluents, substrates and solutions are used, and the assay is run as summarized in the Assay Procedure provided.
Product Features
- Optimized capture and detection antibody pairings with recommended concentrations save lengthy development time
- Development protocols are provided to guide further assay optimization
- Assay can be customized to your specific needs
- Economical alternative to complete kits
Kit Content
- Capture Antibody
- Detection Antibody
- Recombinant Standard
- Streptavidin conjugated to horseradish-peroxidase (Streptavidin-HRP)
Other Reagents Required
DuoSet Ancillary Reagent Kit 2 (5 plates): (Catalog # DY008B) containing 96 well microplates, plate sealers, substrate solution, stop solution, plate coating buffer (PBS), wash buffer, and Reagent Diluent Concentrate 2.
The components listed above may be purchased separately:
PBS: (Catalog # DY006), or 137 mM NaCl, 2.7 mM KCl, 8.1 mM Na2HPO4, 1.5 mM KH2PO4, pH 7.2 - 7.4, 0.2 µm filtered
Wash Buffer: (Catalog # WA126), or equivalent
Reagent Diluent*
Blocking Buffer*
Substrate Solution: ELISA TMB Substrate (R&D Systems,
Catalog # DY999B)
Stop Solution: 2N H2SO4 (Catalog # DY994)
Microplates: R&D Systems (Catalog # DY990)
Plate Sealers: ELISA Plate Sealers (Catalog # DY992)
*For the Reagent Diluent and Blocking Buffer are recommended for a specific DuoSet ELISA Development Kit, see the product datasheet.
Scientific Data
Product Datasheets
Preparation and Storage
Background: OSMR beta
Oncostatin M (OSM) is a cytokine originally isolated from medium conditioned by PMA-treated U-937 human histiocytic leukemia cells based on its ability to inhibit growth of A375 melanoma cells. The human OSM cDNA encodes a 252 amino acid pre-pro-OSM polypeptide with a 25 residue hydrophobic signal peptide and a hydrophilic C-terminal domain that are proteolytically processed to generate the 196 residue mature form of OSM. Although both mature and pro-OSM are equally active in radio-receptor assays, the mature OSM is 5- to 60-fold more active in growth inhibition assays. Thus, proteolytic processing of the pro-OSM peptide may be important in regulating the in vivo activities of OSM. OSM initiates its biological activities by binding to specific cell surface receptors. The gp130, a signal transducing component (beta subunit) of the IL-6, LIF and CNTF receptor complexes, was identified as a low-affinity OSM receptor that does not transduce OSM signals. The low affinity LIF receptor (LIF R, a gp130-related protein) has now been identified to be a component of a high-affinity OSM receptor that will transduce OSM signals. Since OSM is also active on cells that do not express LIF R, a specific OSM receptor that does not involve LIF R must also exist. Besides its growth inhibitory activities on human A375 melanoma and mouse M1 myeloid leukemic cells, as well as on other solid tumor cells, OSM also has growth stimulatory activities on normal fibroblasts, AIDS-Kaposis sarcoma cells, and a human erythroleukemia cell line, TF-1. Other OSM-mediated activities reported to date include: stimulation of plasminogen activator activity in cultured bovine aortic endothelial cells, regulation of IL-6 expression in human endothelial cells, and stimulation of LDL uptake and up-regulation of cell surface LDL receptors in HepG2 cells.
Human Oncostatin M (OSM) signals through two types of human OSM receptor complexes: the type I complex comprising the leukemia inhibitory factor receptor beta (LIF R beta) and gp130 and the type II complex made up of OSM receptor beta (OSM R beta) and gp130. In contrast, mouse OSM signals only through the mouse OSM R beta and gp130 complex. Human and mouse OSM R beta are 55% identical at the amino acid sequence level.
Assay Procedure
GENERAL ELISA PROTOCOL
Plate Preparation
- Dilute the Capture Antibody to the working concentration in PBS without carrier protein. Immediately coat a 96-well microplate with 100 μL per well of the diluted Capture Antibody. Seal the plate and incubate overnight at room temperature.
- Aspirate each well and wash with Wash Buffer, repeating the process two times for a total of three washes. Wash by filling each well with Wash Buffer (400 μL) using a squirt bottle, manifold dispenser, or autowasher. Complete removal of liquid at each step is essential for good performance. After the last wash, remove any remaining Wash Buffer by aspirating or by inverting the plate and blotting it against clean paper towels.
- Block plates by adding 300 μL Reagent Diluent to each well. Incubate at room temperature for a minimum of 1 hour.
- Repeat the aspiration/wash as in step 2. The plates are now ready for sample addition.
Assay Procedure
- Add 100 μL of sample or standards in Reagent Diluent, or an appropriate diluent, per well. Cover with an adhesive strip and incubate 2 hours at room temperature.
- Repeat the aspiration/wash as in step 2 of Plate Preparation.
- Add 100 μL of the Detection Antibody, diluted in Reagent Diluent, to each well. Cover with a new adhesive strip and incubate 2 hours at room temperature.
- Repeat the aspiration/wash as in step 2 of Plate Preparation.
- Add 100 μL of the working dilution of Streptavidin-HRP to each well. Cover the plate and incubate for 20 minutes at room temperature. Avoid placing the plate in direct light.
- Repeat the aspiration/wash as in step 2.
- Add 100 μL of Substrate Solution to each well. Incubate for 20 minutes at room temperature. Avoid placing the plate in direct light.
- Add 50 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. If wavelength correction is available, set to 540 nm or 570 nm. If wavelength correction is not available, subtract readings at 540 nm or 570 nm from the readings at 450 nm. This subtraction will correct for optical imperfections in the plate. Readings made directly at 450 nm without correction may be higher and less accurate.
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