Mouse Osteopontin 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.
This DuoSet ELISA Development kit contains the basic components required for the development of sandwich ELISAs to measure natural and recombinant mouse Osteopontin (OPN). The suggested diluent is suitable for the analysis of most cell culture supernate samples. Diluents for complex matrices, such as serum and plasma, should be evaluated prior to use in this DuoSet.
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 # DY008) 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 0.05% Tween® 20 in PBS, pH 7.2-7.4
Reagent Diluent: (Catalog # DY995), or 1% BSA in PBS, pH 7.2-7.4, 0.2 µm filtered
Substrate Solution: 1:1 mixture of Color Reagent A (H2O2) and Color Reagent B (Tetramethylbenzidine) (Catalog # DY999)
Stop Solution: 2 N H2SO4 (Catalog # DY994)
Microplates: R&D Systems (Catalog # DY990)
Plate Sealers: ELISA Plate Sealers (Catalog # DY992)
Scientific Data
Product Datasheets
Preparation and Storage
Background: Osteopontin/OPN
Osteopontin (OPN), also known as bone sialoprotein (BSP), is a secreted SIBLING family protein that can be variably modified by O- and N-glycosylation, sulfation, phosphorylation, and transglutamination. OPN is widely expressed and is prominent in mineralized tissues. It inhibits bone mineralization and kidney stone formation and promotes inflammation, cell ad¬hesion, and migration. Its expression is upregulat¬ed during inflammation, obesity, atherosclerosis, cancer, and tissue damage and contributes to the pathophysiology of these conditions. The central region of OPN contains RGD and non-RGD binding sites for multiple integrins. Adjacent to the RGD motif is the sequence SLAYGLR (SVVYGLR in human) which serves as a cryptic binding site for additional integrins: it is masked in full length OPN but is exposed following OPN cleavage by multiple proteases in tumors and sites of tissue injury.
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.
Citations for Mouse Osteopontin DuoSet ELISA
R&D Systems personnel manually curate a database that contains references using R&D Systems products. The data collected includes not only links to publications in PubMed, but also provides information about sample types, species, and experimental conditions.
15
Citations: Showing 1 - 10
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Intracellular osteopontin protects from autoimmunity-driven lymphoma development inhibiting TLR9-MYD88-STAT3 signaling
Authors: C Rizzello, V Cancila, S Sangaletti, L Botti, C Ratti, M Milani, M Dugo, F Bertoni, C Tripodo, C Chiodoni, MP Colombo
Molecular Cancer, 2022-12-12;21(1):215.
Species: Mouse, Transgenic Mouse
Sample Types: Serum
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cis interaction of CD153 with TCR/CD3 is crucial for the pathogenic activation of senescence-associated T�cells
Authors: Y Fukushima, K Sakamoto, M Matsuda, Y Yoshikai, H Yagita, D Kitamura, M Chihara, N Minato, M Hattori
Cell Reports, 2022-09-20;40(12):111373.
Species: Mouse
Sample Types: Cell Culture Supernates
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Identification of kidney injury released circulating osteopontin as causal agent of respiratory failure
Authors: FZ Khamissi, L Ning, E Kefaloyian, H Dun, A Arthanaris, A Keller, JJ Atkinson, W Li, B Wong, S Dietmann, K Lavine, D Kreisel, A Herrlich
Science Advances, 2022-02-25;8(8):eabm5900.
Species: Mouse
Sample Types: Serum
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STAT6-Dependent Exacerbation of House Dust Mite-Induced Allergic Airway Disease in Mice by Multi-Walled Carbon Nanotubes
Authors: MD Ihrie, KS Duke, KA Shipkowski, DJ You, HY Lee, AJ Taylor-Jus, JC Bonner
NanoImpact, 2021-03-13;22(0):.
Species: Mouse
Sample Types: BALF
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Lipoteichoic Acid Accelerates Bone Healing by Enhancing Osteoblast Differentiation and Inhibiting Osteoclast Activation in a Mouse Model of Femoral Defects
Authors: CC Hu, CH Chang, YM Hsiao, Y Chang, YY Wu, SWN Ueng, MF Chen
Int J Mol Sci, 2020-08-03;21(15):.
Species: Mouse
Sample Types: Cell Culture Supernates, Protein
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The CD153 vaccine is a senotherapeutic option for preventing the accumulation of senescent T cells in mice
Authors: S Yoshida, H Nakagami, H Hayashi, Y Ikeda, J Sun, A Tenma, H Tomioka, T Kawano, M Shimamura, R Morishita, H Rakugi
Nat Commun, 2020-05-18;11(1):2482.
Species: Mouse
Sample Types: Serum
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OC-STAMP promotes osteoclast fusion for pathogenic bone resorption in periodontitis via up-regulation of permissive fusogen CD9
Authors: T Ishii, M Ruiz-Torru, A Ikeda, S Shindo, A Movila, H Mawardi, A Albassam, RA Kayal, AA Al-Dharrab, K Egashira, W Wisitrasam, K Yamamoto, AI Mira, K Sueishi, X Han, MA Taubman, T Miyamoto, T Kawai
FASEB J., 2018-03-13;0(0):fj201701424R.
Species: Mouse
Sample Types: Tissue Homoegenates
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Physiologic Thymic Involution Underlies Age-Dependent Accumulation of Senescence-Associated CD4(+) T Cells
Authors: K Sato, A Kato, M Sekai, Y Hamazaki, N Minato
J. Immunol., 2017-05-24;0(0):.
Species: Mouse
Sample Types: Cell Culture Supernates
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Contribution of engineered nanomaterials physicochemical properties to mast cell degranulation
Authors: MM Johnson, R Mendoza, AJ Raghavendr, R Podila, JM Brown
Sci Rep, 2017-03-06;7(0):43570.
Species: Mouse
Sample Types: Cell Culture Supernates
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Silica-Triggered Autoimmunity in Lupus-Prone Mice Blocked by Docosahexaenoic Acid Consumption
PLoS ONE, 2016-08-11;11(8):e0160622.
Species: Mouse
Sample Types: BALF
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The impairment of osteogenesis in bone sialoprotein (BSP) knockout calvaria cell cultures is cell density dependent.
Authors: Bouet G, Bouleftour W, Juignet L, Linossier M, Thomas M, Vanden-Bossche A, Aubin J, Vico L, Marchat D, Malaval L
PLoS ONE, 2015-02-24;10(2):e0117402.
Species: Mouse
Sample Types: Cell Culture Supernates
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Skeletal development of mice lacking bone sialoprotein (BSP)--impairment of long bone growth and progressive establishment of high trabecular bone mass.
Authors: Bouleftour W, Boudiffa M, Wade-Gueye N, Bouet G, Cardelli M, Laroche N, Vanden-Bossche A, Thomas M, Bonnelye E, Aubin J, Vico L, Lafage-Proust M, Malaval L
PLoS ONE, 2014-05-09;9(5):e95144.
Species: Mouse
Sample Types: Serum
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Vitamin D receptor agonists increase klotho and osteopontin while decreasing aortic calcification in mice with chronic kidney disease fed a high phosphate diet.
Authors: Lau, Wei Ling, Leaf, Elizabet, Hu, Ming Cha, Takeno, Marc M, Kuro-o, Makoto, Moe, Orson W, Giachelli, Cecilia
Kidney Int, 2012-08-29;82(12):1261-70.
Species: Mouse
Sample Types: Serum
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Rosiglitazone improves survival and hastens recovery from pancreatic inflammation in obese mice.
Authors: Pini M, Rhodes DH, Castellanos KJ
PLoS ONE, 2012-07-16;7(7):e40944.
Species: Mouse
Sample Types: Plasma
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Phosphate feeding induces arterial medial calcification in uremic mice: role of serum phosphorus, fibroblast growth factor-23, and osteopontin.
Authors: El-Abbadi MM, Pai AS, Leaf EM, Yang HY, Bartley BA, Quan KK, Ingalls CM, Liao HW, Giachelli CM
Kidney Int., 2009-03-25;75(12):1297-307.
Species: Mouse
Sample Types: Serum
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