Human FABP4/A-FABP 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 Fatty Acid-Binding Protein 4 (FABP4). The suggested diluent is suitable for the analysis of most cell culture supernate, serum, and palsma 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
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: FABP4/A-FABP
FABP4 (Fatty Acid Binding Protein 4), also known as A-FABP and aP2, is the predominant FABP found in adipocytes and is often used as a marker for adipocyte differentiation. It is also expressed in macrophages, dendritic cells, and endothelial cells. FABP4 is a key mediator of intracellular fatty acid transport and metabolism in adipose tissue. Its expression is regulated by multiple factors including fatty acids, PPAR gamma agonists, and Insulin, and its levels increase with lipolytic stimulation. It can increase the hydrolytic activity of Hormone-Sensitive Lipase and increase the production of proinflammatory cytokines and chemokines. FABP4 upregulation in adipocytes and macrophages is associated with the development of Insulin resistance, hypertriacylglycerolaemia, and atherosclerosis. Serum levels of FABP4 are elevated in obesity and metabolic syndrome, type 2 diabetes, HIV-associated lipodystrophy, polycystic ovary syndrome, nonalcoholic fatty liver disease, atherosclerosis, and acute ischaemic stroke. FABP4 is also overexpressed in multiple cancer types including ovarian, bladder, glioblastoma, and oral, and it may play a role in tumor progression.
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 FABP4/A-FABP 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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Circulating Concentrations of Cathelicidin Anti-Microbial Peptide (CAMP) Are Increased during Oral Glucose Tolerance Test
Authors: Höpfinger, A;Karrasch, T;Schäffler, A;Schmid, A;
International journal of molecular sciences
Species: Human
Sample Types: Serum
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Dysregulation of adipokines levels among healthy first-degree relatives of type 2 diabetes patients
Authors: Purnamasari, D;Simanjuntak, CK;Tricaesario, C;Tahapary, DL;Harbuwono, DS;Yunir, E;
Heliyon
Species: Human
Sample Types: Serum
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Circulating Levels of Cathelicidin Antimicrobial Peptide (CAMP) Are Affected by Oral Lipid Ingestion
Authors: Höpfinger, A;Karrasch, T;Schäffler, A;Schmid, A;
Nutrients
Species: Human
Sample Types: Serum
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We run human serum at a 1:25 dilution with high precision.
