Mouse uPAR 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 uPAR. 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: uPAR
The urokinase-type Plasminogen Activator Receptor (uPAR) is a GPI-anchored protein that binds to both the inactive and active forms of uPA. This interaction serves to localize and enhance the proteolytic activity of uPA, leading to increased conversion of Plasminogen to Plasmin. uPA also triggers uPAR-mediated signal transduction, activation of protein tyrosine kinases, gene expression, cell adhesion, and chemotaxis. uPAR can additionally regulate cell adhesion by direct binding to Vitronectin and preventing Integrins from binding to their ligands.
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 uPAR 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 - 4
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Senolytic CAR T cells reverse senescence-associated pathologies
Authors: C Amor, J Feucht, J Leibold, YJ Ho, C Zhu, D Alonso-Cur, J Mansilla-S, JA Boyer, X Li, T Giavridis, A Kulick, S Houlihan, E Peerschke, SL Friedman, V Ponomarev, A Piersigill, M Sadelain, SW Lowe
Nature, 2020-06-17;0(0):.
Species: Mouse
Sample Types: Plasma
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Bone marrow-derived immature myeloid cells are a main source of circulating suPAR contributing to proteinuric kidney disease
Nat. Med, 2016-12-12;0(0):.
Species: Mouse
Sample Types: Serum
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Soluble Urokinase Receptors in Focal Segmental Glomerulosclerosis: A Review on the Scientific Point of View
J Immunol Res, 2016-07-18;2016(0):2068691.
Species: Mouse
Sample Types: Serum
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A reassessment of soluble urokinase-type plasminogen activator receptor in glomerular disease.
Authors: Spinale J, Mariani L, Kapoor S, Zhang J, Weyant R, Song P, Wong H, Troost J, Gadegbeku C, Gipson D, Kretzler M, Nihalani D, Holzman L
Kidney Int, 2014-10-29;87(3):564-74.
Species: Mouse
Sample Types: Serum
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Great Duoset kit like most Duosets that I have used. Of the duosets that I have used, this one requires careful wash steps and more attention to detail than usual. Bad washing can produce very high CVs so just take your time. Overall, the kit performs sufficiently and I would buy again.
