Human Pro-BDNF 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 Pro-BDNF. 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: BDNF
BDNF (Brain-Derived Neurotrophic Factor) is a secreted protein that regulates many aspects of neuronal development and function in the nervous system. It is produced as proBDNF in response to neuronal activity or inflammatory stimulation and is then cleaved before associating into a homodimer. It is expressed throughout the nervous system as well as by fibroblasts, megakaryocytes/platelets, and smooth muscle cells. BDNF regulates neural stem cell survival and differentiation, axon/dendrite differentiation, synapse formation and maturation, and refinement of developing circuits. It is also a key regulator of synaptic plasticity and late-phase long-term potentiation. BDNF signals through the TrkB and NGF R/TNFRSF16 receptors. Secreted proBDNF can bind to NGF R/TNFRSF16 and induce long-term depression and cell apoptosis.
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 Pro-BDNF 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.
11
Citations: Showing 1 - 10
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Biomarkers of Depression among Adolescent Girls: BDNF and Epigenetics
Authors: Zwoli?ska, W;Bilska, K;Tarhonska, K;Reszka, E;Skibi?ska, M;Pytli?ska, N;S?opie?, A;Dmitrzak-W?glarz, M;
International journal of molecular sciences
Species: Human hepegivirus
Sample Types: Serum
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The relationship between the brain-derived neurotrophic factor and neurocognitive response to physical exercise in individuals with schizophrenia
Authors: Bang-Kittilsen, G;Egeland, J;Ueland, T;Andersen, E;Bigseth, TT;Holmen, TL;Mordal, J;Holst, R;Engh, JA;
Psychoneuroendocrinology
Species: Human
Sample Types: Platelet-poor Plasma
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alpha7 Nicotinic acetylcholine receptor potentiation downregulates chemotherapy-induced inflammatory overactivation by overlapping intracellular mechanisms
Authors: I Marmouzi, S Myers, DJ Buck, RL Davis, HR Arias
The international journal of biochemistry & cell biology, 2023-03-24;158(0):106405.
Species: Mouse
Sample Types: Tissue Lysates
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BDNF, proBDNF and IGF-1 serum levels in na�ve and medicated subjects with autism
Authors: MLA Robinson-A, B Michalski, B Vidal-Mart, LR Hernández, MW Santiesteb, M Fahnestock
Scientific Reports, 2022-08-12;12(1):13768.
Species: Human
Sample Types: Serum
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BDNF and Pro-BDNF in Amyotrophic Lateral Sclerosis: A New Perspective for Biomarkers of Neurodegeneration
Authors: G Riolo, C Ricci, N De Angelis, C Marzocchi, G Guerrera, G Borsellino, F Giannini, S Battistini
Brain sciences, 2022-05-09;12(5):.
Species: Human
Sample Types: Serum
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ProBDNF as an Indicator of Improvement among Women with Depressive Episodes
Authors: W Zwoli?ska, M Skibinska, A S?opie?, M Dmitrzak-W
Metabolites, 2022-04-16;12(4):.
Species: Human
Sample Types: Serum
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Increased ratio of mature BDNF to precursor-BDNF in patients with major depressive disorder with severe anhedonia
Authors: C Wu, J Lu, S Lu, M Huang, Y Xu
J Psychiatr Res, 2020-05-12;126(0):92-97.
Species: Human
Sample Types: Plasma
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N-Glycosylation is required for secretion of the precursor to brain-derived neurotrophic factor (proBDNF) carrying sulfated LacdiNAc structures
Authors: J Benicky, M Sanda, Z Brnakova K, R Goldman
J. Biol. Chem., 2019-09-26;0(0):.
Species: Rat
Sample Types: Cell Culture Supernates
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Learning and memory deficits and alzheimer's disease-like changes in mice after chronic exposure to microcystin-LR
Authors: J Wang, Y Chen, C Zhang, Z Xiang, J Ding, X Han
J. Hazard. Mater., 2019-03-26;373(0):504-518.
Species: Mouse
Sample Types: Cell Culture Supernates
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The serum protein levels of the tPA-BDNF pathway are implicated in depression and antidepressant treatment
Authors: H Jiang, S Chen, C Li, N Lu, Y Yue, Y Yin, Y Zhang, X Zhi, D Zhang, Y Yuan
Transl Psychiatry, 2017-04-04;7(4):e1079.
Species: Human
Sample Types: Serum
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Serum levels of neurotrophic factors in active toxoplasmic retinochoroiditis
Authors: Cynthia Azeredo Cordeiro
Braz J Infect Dis, 2016-12-05;0(0):.
Species: Human
Sample Types: Serum
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Used in EDTA Plasma diluted 1:10 in Reagent Diluent. Replicates are very good, samples well into detection range.
