Bdnf emax immunoassay system

Manufactured by Promega

Sourced in United States, United Kingdom

The BDNF Emax ImmunoAssay System is a quantitative sandwich enzyme-linked immunosorbent assay (ELISA) designed for the measurement of brain-derived neurotrophic factor (BDNF) levels in biological samples. The system provides a standardized and reliable method for the detection and quantification of BDNF concentrations.

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Spelling variants of this product

Emax ImmunoAssay System (31 citations) BDNF Emax ImmunoAssay (9 citations) BDNF Emax ImmunoAssay System kit (8 citations) ELISA BDNF Emax Immunoassay System (5 citations) BDNF Emax (4 citations)

97 protocols using bdnf emax immunoassay system

BDNF ELISA Protocol for Cell Lysates

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Immediately following removal of the culture medium, NG cultures were treated with pre-chilled lysis buffer (20 mM Tris buffer, pH 7.4, 137 mM NaCl, 1% Nonidet-P40, 10% glycerol, 1 mM PMSF, 0.5 mM sodium vanadate, 10 μM aprotinin, 10 μM actinonin, and 100 μM leupeptin; 75 μl/well). Next, the cells were scraped off and the entire well content was transferred to a siliconized (Sigmacote^®; Sigma) and pre-chilled 1.5-ml microcentifuge tube. Each well was then rinsed with 150 μl of pre-chilled Block & Sample buffer (1x; BDNF E_max™ ImmunoAssay System, Promega), and the well content was transferred into the microcentrifuge tube to combine with the cell lysate. The content of two identically treated sister culture wells was combined in one microcentrifuge tube, and each sample (the total volume of 450 μl) was sonicated on ice using a microprobe sonicator (2 × 1.5 W, 5 s each; Sonicator 3000, Misonix, Inc., Farmingdale, NY). Subsequently, the samples were transferred to an anti-BDNF-coated 96-well ELISA plate (4 wells/sample). BDNF ELISA was performed according to the manufacturer’s instructions (BDNF E_max™ ImmunoAssay System, Promega). We have previously confirmed the specificity of the BDNF ELISA kit in our model using RNA interference (Hsieh et al., 2010 (link)).

Vermehren-Schmaedick A., Khanjian R.A, & Balkowiec A. (2015). CELLULAR MECHANISMS OF ACTIVITY-DEPENDENT BDNF EXPRESSION IN PRIMARY SENSORY NEURONS. Neuroscience, 310, 665-673.

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Quantitative BDNF Immunoassay Protocol

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We will use the Promega BDNF Emax ImmunoAssay System and will process the samples and assay serum BDNF levels according to the manufacturer protocols. In short, specimens will be aliquoted into a 96 well plate pre-coated with anti-BDNF monoclonal antibody. BDNF polyclonal antibody will then be added to each well and bind to the bound BDNF. Anti-IgY antibody will be added to each well as a tertiary reactant. Finally, a chromogenic agent will be added to the wells and the degree of color change measured. The amount of BDNF is proportional to the degree of color change detected. Each plate contains controls that prepare a standard curve. The Promega BDNF Emax ImmunoAssay System produces less than 3% cross-reactivity with other neurotrophic factors and can detect BDNF at concentrations as low as 15.6pg/mL. The protocol and procedures have been previously published (BDNF Emax ImmunoAssay System, 2009 ; Pressler et al., 2017 (link)).

Breazeale S., Dorsey S.G., Kearney J., Conley S., Jeon S., Yoo B, & Redeker N.S. (2020). Symptom Cluster Profiles Following Traumatic Orthopedic Injuries: A Protocol. Research in nursing & health, 44(2), 268-278.

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Quantifying Sciatic Nerve BDNF Levels

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BDNF protein levels of sciatic nerve and DRG were analysed using the BDNF Emax^® ImmunoAssay System (Promega, Madison, WI, USA) according to the manufacturer’s protocol as described previously [40 (link)]. Briefly, 96-well plates were coated with anti-BDNF monoclonal antibody and incubated at 4°C overnight. The plates were washed with TBS-T and incubated with 200 μl of Block and Sample buffer (BDNF E_max™ ImmunoAssay System, Promega, Madison, WI, USA) for 1 h at room temperature. 100 μl of each sample prepared as described above was transferred to the anti-BDNF-coated ELISA plate. BDNF levels were determined from the standard curve prepared for each plate. The standard curves were linear within the range used (0–500 pg/ml).

Neumann E., Hermanns H., Barthel F., Werdehausen R, & Brandenburger T. (2015). Expression changes of microRNA-1 and its targets Connexin 43 and brain-derived neurotrophic factor in the peripheral nervous system of chronic neuropathic rats. Molecular Pain, 11, 39.

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Quantification of BDNF and Phospho-TrkB

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BDNF protein concentration in brain lysates was measured with the BDNF Emax® ImmunoAssay System (Promega) according to manufacturer’s instructions. The levels of phosphorylated TrkB were measured by sandwich ELISA assay using the monoclonal anti-phosphotyrosine (clone 4G10, #05-321, Merck-Millipore) and the polyclonal anti-TrkB (#07-225, Merck-Millipore) as capture and detection antibodies, respectively. Anti-rabbit immunoglobulin G coupled to horseradish peroxidase (#31460, Thermo Fisher Scientific) was used as secondary antibody. After incubation with TMB One solution (Promega) absorbance at 450 nm was measured on a Tecan Infinite F500 plate reader.

Almacellas-Barbanoj A., Albini M., Satapathy A., Jaudon F., Michetti C., Krawczun-Rygmaczewska A., Huang H., Manago F., Papaleo F., Benfenati F, & Cesca F. (2022). Kidins220/ARMS modulates brain morphology and anxiety-like traits in adult mice. Cell Death Discovery, 8, 58.

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Quantification of BDNF Levels in Brain Tissue

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Dissected brain tissue (cortex, hippocampus, cerebellum) was collected, stored at −80°C, was thawed and homogenized by pestle in RIPA buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 2 mM EDTA, Teknova) with protease inhibitor cocktail (104 mM AEBSF, 80 μM Aprotinin, 4 mM Bestatin, 1.4 mM E-64, 2 mM Leupeptin, and 1.5 mM Pepstatin A, Sigma). The homogenized tissue was left on ice for 15 min, and then centrifuged at 15,000 RCF for 15 min at 4°C. The supernatant was retained and its protein quantitated by a BSA protein assay (Pierce, ThermoFisher). The samples were acid treated with addition of HCl to a pH of 2–3 for 15 min, then neutralized with NaOH. BDNF content was assayed by a sandwich enzyme-linked immunosorbent assay (ELISA) (BDNF Emax ImmunoAssay System, Promega), utilizing a monoclonal anti-BDNF antibody for plate coating, and a human polyclonal anti-BDNF antibody with an anti-IgY HRP conjugate for colorimetric detection. A BDNF protein standard curve, performed in duplicate, was included on all plates. All samples were assayed in triplicate and then averaged. BDNF protein was quantified relative to total protein.

Kotloski R.J., Rutecki P.A, & Sutula T.P. (2020). Genetic Background Influences Acute Response to TBI in Kindling-Susceptible, Kindling-Resistant, and Outbred Rats. Frontiers in Neurology, 10, 1286.

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Quantifying BDNF Levels in NS-1 Cells

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After MS, NS-1 cells were homogenized in lysis buffer 100mM PIPES (pH 7), 500mM NaCl, 0.2% Triton X-100, 0.1% NaN₃, 2% BSA, 2mM EDTA·Na₂·2H₂O (Szapacs et al., 2004 (link)) containing complete protease inhibitor cocktail (Roche, South San Francisco, CA). Sample protein content was measured (BioRad Protein Assay: BioRad Laboratories, Hercules, CA) and volumes were adjusted to normalize for protein content. Aliquots were then processed for BDNF ELISA using the BDNF Emax Immunoassay System (Promega, Madison, WI) as described by the manufacturer. To measure total BDNF, cell lysates were acid-treated and then neutralized before proceeding with ELISA. To measure mature BDNF (mBDNF), the ELISA protocol was performed without acid treatment. The BDNF levels were determined relative to a standard curve constructed from measures of the kit-supplied BDNF protein standards (0–500 pg BDNF protein) that were assayed simultaneously with experimental samples. Data are presented as means±SEM pg BDNF/μg of sample protein content.

Lin C.Y., Huang W.J., Li K., Swanson R., Cheung B., Lin V.W, & Lee Y.S. (2015). Differential Intensity-dependent Effects of Magnetic Stimulation on the Longest Neurites and Shorter Dendrites in Neuroscreen-1 Cells. Journal of neural engineering, 12(2), 026013.

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Quantifying U87 Cell Secretions

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NGF, BDNF, and IL-6 secreted from cultured U87 cells in the presence of peptides were determined using the NGF Emax ImmunoAssay System (Promega, MA, USA), BDNF Emax ImmunoAssay System (Promega, MA, USA), and BD Opt EIA Human IL-6 ELISA Set (BD Pharmingen, CA, USA) according to the procedures provided by the manufacturer.

Zabłocka A., Mitkiewicz M., Macała J, & Janusz M. (2015). Neurotrophic Activity of Cultured Cell Line U87 is Up-Regulated by Proline-Rich Polypeptide Complex and Its Constituent Nonapeptide. Cellular and Molecular Neurobiology, 35(7), 977-986.

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Measuring Serum BDNF Protein Levels

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Venous blood was drawn from each participant in the morning following a 6-hour fast. Levels of fasting blood glucose, triglyceride (TG), high-density lipoprotein cholesterol (HDL-C), and total cholesterol were measured in the hospital laboratory. Serum BDNF protein levels were measured using a commercially available enzyme-linked-immunosorbent serologic assay kit of the sandwich type (BDNF Emax Immunoassay System; Promega, Madison, WI, USA). Each system contained anti-BDNF mAb, Block&Sample 5× buffer, BDNF standard, antihuman BDNF pAb, anti-IgY HRP, TMB solution, peroxidase substrate, and protocol. All samples were assayed or duplicated by the same senior laboratory assistant.

Lin C.C., Hung Y.Y., Tsai M.C, & Huang T.L. (2017). Increased serum brain-derived neurotrophic factor in male schizophrenic patients with metabolic syndrome. Medicine, 96(22), e7089.

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Measuring Inflammatory and Neurotropic Factors

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Serum Interleukin-6 (IL-6) levels were assessed at sacrifice (72 hours and 4 weeks) in both cohorts of mice according to manufacturer’s instructions (eBiosciences, San Diego, CA). Total BDNF was measure by using BDNF Emax^® ImmunoAssay System by Promega following their protocol.

Verma R., Harris N.M., Friedler B.D., Crapser J., Patel A.R., Venna V, & McCullough L.D. (2016). Reversal of the Detrimental Effects of Post-Stroke Social Isolation by Pair-Housing is Mediated by Activation of BDNF-MAPK/ERK in Aged Mice. Scientific Reports, 6, 25176.

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BDNF Measurement Using Sandwich ELISA

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Brain-derived neurotrophic factor was measured with a conventional sandwich ELISA using the BDNF Emax Immuno-Assay System (Promega, Madison, WI, USA) according to the protocol of the manufacturer.

Pandya C.D, & Pillai A. (2014). TrkB interacts with ErbB4 and regulates NRG1-induced NR2B phosphorylation in cortical neurons before synaptogenesis. Cell Communication and Signaling : CCS, 12, 47.

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