Human Thrombospondin-1 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 human Thrombospondin-1. 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: Thrombospondin-1
Thrombospondins (TSP) are secreted multidomain glycoproteins with many putative functions including modulating cell adhesion, proliferation, migration, and angiogenesis. At least five TSPs exist: TSP-1, -2, -3, -4, and cartilage oligomeric glycoprotein (COMP)/TSP-5. The family can be divided into two subgroups. The first, TSP-1 and -2, are homotrimeric molecules, each monomer containing an N-terminal/heparin-binding domain, a coiled-coil oligomerization domain, a von Willebrand factor-type/procollagen homology domain, three type 1 thrombospondin repeats (TSR), three EGF-like repeats, seven TSP type 3 repeats, and a C-terminal globular domain. TSP-1 and -2 are thought to mediate interactions between cells and the extracellular matrix, and as such are matricellular proteins. In contrast, TSP-3, -4, and COMP/TSP-5 are homopentamers that lack the N-terminal structure of TSP-1 and -2, but have a similar arrangement of EGF and TSP type 3 repeats, and the C-terminal domain characteristic of the TSP family.
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 Human Thrombospondin-1 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.
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Citations: Showing 1 - 10
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Baseline circulating biomarkers, their changes, and subsequent suicidal ideation and depression severity at 6 months: A prospective analysis in patients with mood disorders
Authors: Lengvenyte, A;Cognasse, F;Hamzeh-Cognasse, H;Sénèque, M;Strumila, R;Olié, E;Courtet, P;
Psychoneuroendocrinology
Species: Human
Sample Types: Plasma
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In Vitro Comparison of Lymphangiogenic Potential of Hypoxia Preconditioned Serum (HPS) and Platelet-Rich Plasma (PRP)
Authors: J Jiang, X Cong, S Alageel, U Dornseifer, AF Schilling, E Hadjipanay, HG Machens, P Moog
International Journal of Molecular Sciences, 2023-01-19;24(3):.
Species: Human
Sample Types: Hypoxia Preconditioned Serum
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Pro-cancerogenic effects of spontaneous and drug-induced senescence of ovarian cancer cells in vitro and in vivo: a comparative analysis
Authors: S Rutecki, P Szulc, M Paku?a, P Uruski, A Radziemski, E Naumowicz, R Moszy?ski, A Tykarski, J Miku?a-Pie, K Ksi??ek
Journal of ovarian research, 2022-07-26;15(1):87.
Species: Human
Sample Types: Cell Culture Supernates
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Heme stimulates platelet mitochondrial oxidant production to induce targeted granule secretion
Authors: GK Annarapu, D Nolfi-Done, M Reynolds, Y Wang, L Kohut, B Zuckerbrau, S Shiva
Redox Biology, 2021-12-05;48(0):102205.
Species: Human
Sample Types: Cell Culture Supernates
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Regional hyperthermia enhances mesenchymal stem cell recruitment to tumor stroma: Implications for mesenchymal stem cell-based tumor therapy
Authors: M Tutter, C Schug, KA Schmohl, S Urnauer, C Kitzberger, N Schwenk, M Petrini, C Zach, S Ziegler, P Bartenstei, WA Weber, G Multhoff, E Wagner, LH Lindner, PJ Nelson, C Spitzweg
Mol Ther, 2020-10-15;0(0):.
Species: Human
Sample Types: Cell Culture Supernates
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The role of EMMPRIN/CD147 in regulating angiogenesis in patients with psoriatic arthritis
Authors: MA Rahat, M Safieh, E Simanovich, E Pasand, T Gazitt, A Haddad, M Elias, D Zisman
Arthritis Res Ther, 2020-10-14;22(1):240.
Species: Human
Sample Types: Serum
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Mesenchymal Stem and Stromal Cells Harness Macrophage-Derived Amphiregulin to Maintain Tissue Homeostasis
Authors: JH Ko, HJ Kim, HJ Jeong, HJ Lee, JY Oh
Cell Rep, 2020-03-17;30(11):3806-3820.e6.
Species: Human
Sample Types: Cell Culture Supernates
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Endothelial MT1-MMP targeting limits intussusceptive angiogenesis and colitis via TSP1/nitric oxide axis
Authors: S Esteban, C Clemente, A Koziol, P Gonzalo, C Rius, F Martínez, PM Linares, M Chaparro, A Urzainqui, V Andrés, M Seiki, JP Gisbert, AG Arroyo
EMBO Mol Med, 2019-12-03;0(0):e10862.
Species: Human
Sample Types: Serum
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Thrombospondin-1 secreted by human umbilical cord blood-derived mesenchymal stem cells rescues neurons from synaptic dysfunction in Alzheimer's disease model.
Authors: Dong Hyun Kim, Hoon Lim, Dahm Lee, Soo Jin Choi, Wonil Oh, Yoon Sun Yang, Jeong Su Oh, Hyun Ho Hwang, Hong Bae Jeon
Scientific Reports, 2018-01-10;0(0):2045-2322.
Species: Human
Sample Types: Cell Culture Supernates
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A combination of platelet features allows detection of early-stage cancer
Authors: S Sabrkhany, MJE Kuijpers, SMJ van Kuijk, L Sanders, S Pineda, SWM Olde Damin, AC Dingemans, AW Griffioen, MGA Oude Egbri
Eur. J. Cancer, 2017-05-17;80(0):5-13.
Species: Human
Sample Types: Platelet Poor Plasma
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microRNAs expression in endometriosis and their relation to angiogenic factors.
Authors: Ramon LA, Braza-Boils A, Gilabert-Estelles J, Gilabert J, Espana F, Chirivella M, Estelles A
Hum. Reprod., 2011-02-18;26(5):1082-90.
Species: Human
Sample Types: Tissue Homogenates
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