Human Gas1 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 Gas1. 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: Gas1
The growth arrest-specific genes are related by their induction following cellular stress. Gas1 is a GPI-linked membrane glycoprotein that blocks entry to S phase cell cycle. Gas1 inhibits cell proliferation, protects endothelial cells from apoptosis, and promotes excitotoxic neuronal death. Gas6 is a vitamin K-dependent protein with a gamma-carboxylated amino terminus (Gla domain), four EGF-like repeats, and C-terminal globulin-like domains. Gas6 is a ligand for the Axl, Dtk, and Mer tyrosine kinase receptors. Gas6 is mitogenic and anti-apoptotic for some cell types and also promotes cell adhesion.
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 Gas1 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 - 3
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Simultaneous Treatment with Soluble Forms of GAS1 and PTEN Reduces Invasiveness and Induces Death of Pancreatic Cancer Cells
Authors: L Daniel-Gar, P Vergara, A Navarrete, RO González, J Segovia
Onco Targets Ther, 2020-11-17;13(0):11769-11779.
Species: Human
Sample Types: Cell Culture Supernates
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The p38-MK2/3 Module Is Critical for IL-33-Induced Signaling and Cytokine Production in Dendritic Cells
Authors: C Göpfert, N Andreas, F Weber, N Häfner, T Yakovleva, M Gaestel, T Kamradt, S Drube
J. Immunol., 2017-12-29;0(0):.
Species: Mouse
Sample Types: Cell Culture Supernates
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Growth arrest-specific protein 1 is a novel endogenous inhibitor of glomerular cell activation and proliferation.
Authors: van Roeyen C, Zok S, Pruessmeyer J, Boor P, Nagayama Y, Fleckenstein S, Cohen C, Eitner F, Grone H, Ostendorf T, Ludwig A, Floege J
Kidney Int, 2012-12-19;83(2):251-63.
Species: Human
Sample Types: Cell Culture Supernates
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It is very useful to quantifie the levels of soluble Human Gas1 in cell culture media. This kit is very useful to recognizes the Human Gas1 protein in non-reducing conditions. In the article, "The Internalization of Neurotensin by the Low-Affinity Neurotensin Receptors (NTSR2 and vNTSR2) Activates ERK 1/2 in Glioma Cells and Allows Neurotensin Polyplex Transfection of tGAS1;DOI 10.1007/s10571-015-0172-z", it was demonstrated that the antibody only recognized a recombinat soluble version of human Gas1 in transfected cells.
