Human UCH-L1/PGP9.5 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.
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
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)
Product Datasheets
Preparation and Storage
Background: UCH-L1/PGP9.5
Ubiquitin Carboxyl-terminal Esterase L1 (UCH-L1), also known as PGP9.5, is a deubiquitinating enzyme with a predicted molecular weight of 25 kDa. The human protein shares 95% amino acid sequence identity with its mouse and rat orthologs. UCH-L1 is expressed abundantly in neurons, accounting for 1-2% of total soluble proteins in the brain. It localizes primarily to the cytoplasm, but a subpopulation has been shown to be transiently nuclear. UCH-L1 contains two catalytic residues, Cys90 and His161, which are required for isopeptide bond cleavage at the C-terminal glycine residue of Ubiquitin. The levels of free Ubiquitin appear to be partially regulated by UCH-L1 through the hydrolysis of small Ubiquitin chains and the stabilization of monomeric Ubiquitin. Mice lacking functional UCH-L1 show neuronal dysfunction and neurodegeneration, and mutations in this enzyme have been linked to Parkinson's disease, suggesting that it is important for proper central nervous system function. UCH-L1 also likely plays a complex role in cancer. It has been reported to function as an oncogene in lymphoma, colorectal cancer, and nonsmall cell lung carcinoma. In contrast, it is thought to function as a tumor suppressor protein in nasopharyngeal and breast cancers.
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 UCH-L1/PGP9.5 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 - 2
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Neurobiomarker and body temperature responses to recreational marathon running
Authors: Stacey, MJ;Leckie, T;Fitzpatrick, D;Hodgson, L;Barden, A;Jenkins, R;Galloway, R;Weller, C;Grivas, GV;Pitsiladis, Y;Richardson, AJ;Woods, DR;
Journal of science and medicine in sport
Species: Human
Sample Types: Plasma
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Distribution of five clinically important neuroglial proteins in the human brain
Authors: K Sjölin, K Kultima, A Larsson, E Freyhult, C Zjukovskaj, K Alkass, J Burman
Molecular Brain, 2022-06-29;15(1):52.
Species: Human
Sample Types: Tissue Homogenates
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