Human u-Plasminogen Activator/Urokinase DuoSet ELISA
Discontinued Product
Human u-Plasminogen Activator/Urokinase 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 uPA/Urokinase. 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: u-Plasminogen Activator (uPA)/Urokinase
u-Plasminogen Activator (uPA) is a serine protease that converts plasminogen to plasmin, with roles in a variety of normal and pathological processes that include cell migration and tissue destruction. uPA is a potent marker of invasion and metastasis in a variety of human cancers including breast, stomach, colon, bladder, ovarian, brain, and endometrium.
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 u-Plasminogen Activator/Urokinase 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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Mast Cell Proteases Promote Diverse Effects on the Plasminogen Activation System and Wound Healing in A549 Alveolar Epithelial Cells
Authors: S Mogren, F Berlin, L Eskilsson, N Van Der Bu, E Tufvesson, CK Andersson
Cells, 2022-09-18;11(18):.
Species: Human
Sample Types: BALF
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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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Antibody microarray analysis of amniotic fluid proteomes in women with cervical insufficiency and short cervix, and their association with pregnancy latency length
Authors: S Hong, KH Park, YE Lee, JE Lee, YM Kim, E Joo, I Cho
PLoS ONE, 2022-02-07;17(2):e0263586.
Species: Human
Sample Types: Amniotic Fluid
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bFGF stimulated plasminogen activation factors, but inhibited alkaline phosphatase and SPARC in stem cells from apical Papilla: Involvement of MEK/ERK, TAK1 and p38 signaling
Authors: MC Chang, NY Chen, JH Chen, WL Huang, CY Chen, CC Huang, YH Pan, HH Chang, JH Jeng
Oncogene, 2021-12-28;40(0):95-107.
Species: Human
Sample Types: Cell Culture Supernates
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Changes in liver steatosis in HIV-positive women are associated with the BMI, but not with biomarkers
Authors: R Fernandez-, MW Plankey, D Ware, J Bordon
Cytokine, 2021-05-12;0(0):155573.
Species: Human
Sample Types: Plasma
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A Unique Pattern of Mesothelial-Mesenchymal Transition Induced in the Normal Peritoneal Mesothelium by High-Grade Serous Ovarian Cancer
Authors: M Paku?a, P Uruski, A Niklas, A Wo?niak, D Szpurek, A Tykarski, J Miku?a-Pie, K Ksi??ek
Cancers (Basel), 2019-05-13;11(5):.
Species: Human
Sample Types: Cell Culture Supernates
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Serum from Varicose Patients Induces Senescence-Related Dysfunction of Vascular Endothelium Generating Local and Systemic Proinflammatory Conditions
Oxid Med Cell Longev, 2016-11-23;2016(0):2069290.
Species: Human
Sample Types: Cell Culture Supernates
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Stand-Sit Microchip for High-Throughput, Multiplexed Analysis of Single Cancer Cells
Sci Rep, 2016-09-01;6(0):32505.
Applications: ChIP -
Gelsolin induces colorectal tumor cell invasion via modulation of the urokinase-type plasminogen activator cascade.
Authors: Zhuo, Jingli, Tan, Ee Hong, Yan, Benedict, Tochhawng, Lalchhan, Jayapal, Manikand, Koh, Shiuan, Tay, Hwee Kee, Maciver, Sutherla, Hooi, Shing Ch, Salto-Tellez, Manuel, Kumar, Alan Pre, Goh, Yaw Chon, Lim, Yaw Chyn, Yap, Celestia
PLoS ONE, 2012-08-21;7(8):e43594.
Species: Human
Sample Types: Cell Culture Supernates
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Cerebrospinal fluid u-plasminogen activator and matrix metalloproteinase-9 levels in human eosinophilic meningitis associated with angiostrongyliasis.
Authors: Sanpool O, Intapan PM, Thanchomnang T
Cytokine, 2010-06-26;51(3):294-7.
Species: Human
Sample Types: CSF
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