ProBDNF [30 (link)] was measured in placental homogenates using the BDNF Emax ImmunoAssay acid treatment protocol (Promega) to facilitate dissociation of BDNF from TRKB, thus improving detection. BDNF concentrations were normalized to total protein amounts.
Bdnf emax immunoassay
Manufactured by Promega
Sourced in United States
The BDNF Emax ImmunoAssay is a quantitative sandwich enzyme-linked immunosorbent assay (ELISA) designed to measure the concentration of brain-derived neurotrophic factor (BDNF) in biological samples such as serum, plasma, and cell culture supernatants. The assay is based on a solid-phase enzyme immunoassay technique and provides a simple, accurate, and reproducible method for the quantitative determination of BDNF levels.
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Lab products found in correlation
Microplate reader, by Bio-Rad (3 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Colorimetric assay, by Oxford Biomedical Research (1 mentions)
Blocking buffer, by Promega (1 mentions)
T per buffer, by Thermo Fisher Scientific (1 mentions)
Protease and phosphatase inhibitor cocktail, by Roche (1 mentions)
Pd98059, by Merck Group (1 mentions)
Ly294002, by Merck Group (1 mentions)
Enzyme linked immunosorbent assay kit, by MyBioSource (1 mentions)
9 protocols using bdnf emax immunoassay
1
Measuring Placental ProBDNF Levels
2
ALA Supplementation Increases BDNF Levels
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This study was evaluated and approved by the Ethical Committee of the Tehran University of Medical Sciences. Thirty healthy volunteers, fifteen men and fifteen women, were selected randomly. They read and signed a consent form prior to enrolment in this study. These individuals had Body Mass Indexes (BMI) of less than thirty, similar low-fat diets, and no underlying diseases such as diabetes or high blood pressure. Because effective doses of ALA for increasing BDNF levels are unknown, each individual served as his or her own control.
Before consuming the Flaxseed oil [(Swiss, Canada) (Table1 )], 5cc blood from each individual was sampled in order to measure the plasma levels of BDNF and MDA as baseline controls. During the experiment, each individual was given 3 oral capsules of flaxseed oil, containing 500mg of ALA, daily for one week. Then, Plasma levels of BDNF and MDA were assessed using BDNF Emax® ImmunoAssay (Promega) and colorimetric Assay (Oxford Biomedical Research) kits according to the manufacturer’s protocols, respectively.
Before consuming the Flaxseed oil [(Swiss, Canada) (Table
| Flax seed oil | ALA | Company |
|---|---|---|
| 1000 mg | 530 mg | Swissherbal |
Each 1000 mg flax seed oil capsule contained 530 mg ALA.
3
Quantification of BDNF Protein via ELISA
4
Measurement of BDNF Levels in Cortical Cells
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Supernatant was collected from cortical cell cultures. Cortical cells where lysed using T-PER buffer (Thermo scientific) containing protease and phosphatase inhibitor cocktail (Roche). Cell extracts were obtained after centrifugation at 14000g for 10 minutes. Protein concentrations from each sample were then measured using a Pierce BCA protein assay kit (Thermo scientific) and normalized. Thus, BDNF levels were measured in cellular media and cortical cellular extracts using an ELISA kit according to the manufacturer’s instructions (BDNF Emax Immunoassay (Promega Corp, Madison, WI). Briefly, the monoclonal antibody was added to each well of a 96-well plate and incubated overnight at 4°C. The following were added to the wells sequentially: samples and BDNF standards in duplicate (incubated 2 hours at room temperature), anti-human BDNF pAb (incubated 2 hours at room temperature), anti-IgY HRP (incubated 1 hour at room temperature), and TMB solution (incubated 20 minutes at room temperature). The plate was washed with Tris-buffered saline 0.05% Tween 20 buffer. Finally, a stop solution was added, and absorbance was read at 450nm using a microplate reader (Bio-Rad Laboratories) within 30 minutes of the addition of the stop solution. BDNF concentration was calculated based on a standard curve. Values are expressed in pg/mL protein.
5
BDNF and NGF Quantification Assay
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BDNF concentration was determined using the BDNF Emax ImmunoAssay (Promega, Madison, WI, USA) according to the manufacturer’s instructions. First, a 96-well microplate was sealed, incubated with anti-BDNF antibody (1:1000) overnight at 4°C, and washed with Tris-buffered saline (TBS). On the next day, the plate was blocked at RT for 1 h and washed. Samples and BDNF standards were added into the plate and incubated at RT for 2 h. After an extensive wash, the anti-BDNF antibody (1:500) was added to each well and the plate was incubated at RT for 2 h. After an additional wash, the HRP-conjugated anti-IgY (1:200) was added. One hour later, the plate was washed and incubated with TMB One solution for 10 min at RT. The reaction was ended by adding 1 M HCl. The absorbance was measured at 450 nm. The same procedures were used to determine the level of nerve growth factor (NGF).
6
Astrocyte BDNF Production: MK-801 and Inhibitors
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Cultured astrocytes were passaged and plated in 3.5 cm dishes at 1 × 106 cells/dish, then either incubated directly in 20 μM MK-801 for 24 h or first pretreated with 20 μM PD98059 (Sigma), 20 μM LY294002 (Sigma), 20 μM SP600125, or 20 μM SB203580 for 2 h prior to incubation in MK-801. The culture supernatants were collected and BDNF concentrations measured using a commercially available ELISA kit (BDNF Emax Immunoassay; Promega, Madison, WI, United States), according to the manufacturer’s instructions. Briefly, a monoclonal antibody was added to each well of 96-well plates followed by overnight incubation at 4°C. The following reagents were then sequentially added to the wells: samples and BDNF standards in duplicate (2 h at room temperature), anti-human BDNF polyclonal antibody (2 h at room temperature), anti-IgY horseradish peroxidase (1 h at room temperature), and 3,3’,5,5’-tetramethylbenzidine solution (20 min at room temperature). Plates were washed with Tris-buffered saline containing 0.05% Tween 20 and a stop solution was added to each well. The absorbance was measured at 450 nm using a microplate reader (Bio-Rad) within 30 min. BDNF concentration was calculated based on a standard curve.
7
Quantifying BDNF Levels in Mouse Brain
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BDNF protein levels in the striatum and the PFC were determined 1 week after the last dyskinesia measurement. Levodopa and nicotine treatments were continued until the mice were killed by cervical dislocation 4 h after the last levodopa administration and the brain dissected on ice. Samples were stored at -80 °C until use. Extraction of proteins was done essentially as previously described [47 (link)]. A commercial ELISA kit (BDNF Emax ImmunoAssay; Promega, Madison, WI, USA) was used for the BDNF analyses. The manufacturer’s instructions were followed except for two modifications. First, Optacoat (ALerCHEK, Springvale, ME, USA) was used as the coating buffer, and second, the samples were transiently acidified and neutralized [47 (link)] without first diluting them in DPBS, followed by dilution with Block and Sample 1X Buffer.
8
BDNF Quantification in Cell Cultures
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BDNF concentration in the culture supernatant was measured using a commercially available enzyme-linked immunosorbent assay (ELISA) kit (BDNF Emax Immunoassay; Promega, Madison, WI, USA) according to the manufacturer’s instructions. Briefly, the monoclonal antibody was added to each well of a 96-well plate followed by overnight incubation at 4°C. The following reagents were then sequentially added to the wells: samples and BDNF standards in duplicate (with incubation for 2 h at room temperature); anti-human BDNF polyclonal antibody (with incubation for 2 h at room temperature); anti-IgY horseradish peroxidase (HRP; with incubation for 1 h at room temperature); and 3,3′,5,5′-tetramethylbenzidine solution (with incubation for 20 min at room temperature). The plate was washed with Tris-buffered saline (TBS) containing 0.05% Tween 20. A stop solution was added to each well and absorbance was measured at 450 nm with a microplate reader (Bio-Rad, Hercules, CA, USA) within 30 min. BDNF concentration was calculated based on a standard curve.
9
Plasma CORT and BDNF Measurement
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After the completion of the behavioral tests, on Day 17, blood was collected using the restrained tail snip. We chose this method because it causes minimal stress-related changes in plasma CORT levels due to handling (Kim et al., 2018) (link).
Blood collection was performed between 11:30 am and 2 pm to avoid potential effects of circadian variations on plasma CORT concentrations. Each mouse (n=33) was placed in a restriction tube and a small puncture at the level of the lateral tail vein was made using a sterile blade. Blood samples were collected into EDTA tubes, which were stored on ice until centrifugation. To obtain plasma, blood samples were centrifuged for 15 minutes at 1600g at 4 • C. The supernatant (20-80µl) was collected and stored at -80 °C until analysis.
Plasma CORT analysis was performed on all the mice (n=33) while BDNF measurements were done on 31 mice. Due to outliers, we discarded the results of one mouse from BDNF measurements. We performed these measures using enzyme-linked immunosorbent assay (ELISA) kits: Enzyme-linked immunosorbent Assay kit (My BioSource, San Diego, California, USA) and BDNF Emax ® ImmunoAssay (Promega, Madison, Wisconsin, USA) in accordance with the recommendations of the manufacturer.
Blood collection was performed between 11:30 am and 2 pm to avoid potential effects of circadian variations on plasma CORT concentrations. Each mouse (n=33) was placed in a restriction tube and a small puncture at the level of the lateral tail vein was made using a sterile blade. Blood samples were collected into EDTA tubes, which were stored on ice until centrifugation. To obtain plasma, blood samples were centrifuged for 15 minutes at 1600g at 4 • C. The supernatant (20-80µl) was collected and stored at -80 °C until analysis.
Plasma CORT analysis was performed on all the mice (n=33) while BDNF measurements were done on 31 mice. Due to outliers, we discarded the results of one mouse from BDNF measurements. We performed these measures using enzyme-linked immunosorbent assay (ELISA) kits: Enzyme-linked immunosorbent Assay kit (My BioSource, San Diego, California, USA) and BDNF Emax ® ImmunoAssay (Promega, Madison, Wisconsin, USA) in accordance with the recommendations of the manufacturer.
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