BDNF Antibody [A7P4] | Primary Antibodies

Application: Reactivity: Human, Mouse

Lane 1: 293T, Lane 2: 293T (BDNF(human) transfected)

Experiment Essentials

WB
Recommended wet transfer conditions: 200 mA, 60 min.

Usage Information

Dilution
WB1:1000 IP1:50

Application
WB, IP

Reactivity
Human, Mouse

Source
Rabbit Monoclonal Antibody

Storage Buffer
PBS, pH 7.2+50% Glycerol+0.05% BSA+0.01% NaN3

Storage (from the date of receipt)
-20°C (avoid freeze-thaw cycles), 2 years

Predicted MW Observed MW
28 kDa 28 kDa (ProBDNF), 12-14 kDa (Mature)
^*Why do the predicted and actual molecular weights differ? The following reasons may explain differences between the predicted and actual protein molecular weight. Post-translational modifications(e.g., phosphorylation, glycosylation); Splice variants and isoforms; Relative charge; Multimerization.

Experimental Methods

WB
Experimental Protocol: Sample preparation 1. Tissue: Lyse the tissue sample by adding an appropriate volume of ice-cold RIPA/NP-40 Lysis Buffer (containing Protease Inhibitor Cocktail)，and homogenize the tissue at a low temperature or lyse it by sonication on ice, then incubate on ice for 30 minutes. 2. Adherent cell: Aspirate the culture medium and wash the cells with ice-cold PBS twice. Lyse the cells by adding an appropriate volume of RIPA/NP-40 Lysis Buffer (containing Protease Inhibitor Cocktail) , sonicate to lyse the cells, and incubate on ice for 30 minutes. 3. Suspension cell: Transfer the culture medium to a pre-cooled centrifuge tube. Centrifuge and aspirate the supernatant. Wash the cells with ice-cold PBS twice. Lyse the cells by adding an appropriate volume of RIPA/NP-40 Lysis Buffer (containing Protease Inhibitor Cocktail) , sonicate to lyse the cells, and incubate on ice for 30 minutes. 4. Place the lysate into a pre-cooled microcentrifuge tube. Centrifuge at 4°C for 15 min. Collect the supernatant; 5. Remove a small volume of lysate to determine the protein concentration; 6. Combine the lysate with protein loading buffer. Boil 20 µL sample under 95-100°C for 5 min. Centrifuge for 5 min after cool down on ice. Electrophoretic separation 1. According to the concentration of extracted protein, load appropriate amount of protein sample and marker onto SDS-PAGE gels for electrophoresis. Recommended separating gel (lower gel) concentration: 10%. Reference Table for Selecting SDS-PAGE Separation Gel Concentrations 2. Power up 80V for 30 minutes. Then the power supply is adjusted (110 V~150 V), the Marker is observed, and the electrophoresis can be stopped when the indicator band of the predyed protein Marker where the protein is located is properly separated. (Note that the current should not be too large when electrophoresis, too large current (more than 150 mA) will cause the temperature to rise, affecting the result of running glue. If high currents cannot be avoided, an ice bath can be used to cool the bath.) Transfer membrane 1. Take out the converter, soak the clip and consumables in the pre-cooled converter; 2. Activate PVDF membrane with methanol for 1 min and rinse with transfer buffer; 3. Install it in the order of "black edge of clip - sponge - filter paper - filter paper - glue -PVDF membrane - filter paper - filter paper - sponge - white edge of clip"; 4. The protein was electrotransferred to PVDF membrane. ( 0.45 µm PVDF membrane is recommended ) Reference Table for Selecting PVDF Membrane Pore Size Specifications Recommended conditions for wet transfer: 200 mA, 60 min. ( Note that the transfer conditions can be adjusted according to the protein size. For high-molecular-weight proteins, a higher current and longer transfer time are recommended. However, ensure that the transfer tank remains at a low temperature to prevent gel melting.) Block 1. After electrotransfer, wash the film with TBST at room temperature for 5 minutes; 2. Incubate the film in the blocking solution for 1 hour at room temperature; 3. Wash the film with TBST for 3 times, 5 minutes each time. Antibody incubation 1. Use 5% skim milk powder to prepare the primary antibody working liquid (recommended dilution ratio for primary antibody 1:1000), gently shake and incubate with the film at 4°C overnight; 2. Wash the film with TBST 3 times, 5 minutes each time; 3. Add the secondary antibody to the blocking solution and incubate with the film gently at room temperature for 1 hour; 4. After incubation, wash the film with TBST 3 times for 5 minutes each time. Antibody staining 1. Add the prepared ECL luminescent substrate (or select other color developing substrate according to the second antibody) and mix evenly; 2. Incubate with the film for 1 minute, remove excess substrate (keep the film moist), wrap with plastic film, and expose in the imaging system.

WB

Experimental Protocol:

Sample preparation

1. Tissue: Lyse the tissue sample by adding an appropriate volume of ice-cold RIPA/NP-40 Lysis Buffer (containing Protease Inhibitor Cocktail)，and homogenize the tissue at a low temperature or lyse it by sonication on ice, then incubate on ice for 30 minutes.

2. Adherent cell: Aspirate the culture medium and wash the cells with ice-cold PBS twice. Lyse the cells by adding an appropriate volume of RIPA/NP-40 Lysis Buffer (containing Protease Inhibitor Cocktail) , sonicate to lyse the cells, and incubate on ice for 30 minutes.

3. Suspension cell: Transfer the culture medium to a pre-cooled centrifuge tube. Centrifuge and aspirate the supernatant. Wash the cells with ice-cold PBS twice. Lyse the cells by adding an appropriate volume of RIPA/NP-40 Lysis Buffer (containing Protease Inhibitor Cocktail) , sonicate to lyse the cells, and incubate on ice for 30 minutes.

4. Place the lysate into a pre-cooled microcentrifuge tube. Centrifuge at 4°C for 15 min. Collect the supernatant;

5. Remove a small volume of lysate to determine the protein concentration;

6. Combine the lysate with protein loading buffer. Boil 20 µL sample under 95-100°C for 5 min. Centrifuge for 5 min after cool down on ice.

Electrophoretic separation

1. According to the concentration of extracted protein, load appropriate amount of protein sample and marker onto SDS-PAGE gels for electrophoresis. Recommended separating gel (lower gel) concentration: 10%. Reference Table for Selecting SDS-PAGE Separation Gel Concentrations

2. Power up 80V for 30 minutes. Then the power supply is adjusted (110 V~150 V), the Marker is observed, and the electrophoresis can be stopped when the indicator band of the predyed protein Marker where the protein is located is properly separated. (Note that the current should not be too large when electrophoresis, too large current (more than 150 mA) will cause the temperature to rise, affecting the result of running glue. If high currents cannot be avoided, an ice bath can be used to cool the bath.)

Transfer membrane

1. Take out the converter, soak the clip and consumables in the pre-cooled converter;

2. Activate PVDF membrane with methanol for 1 min and rinse with transfer buffer;

3. Install it in the order of "black edge of clip - sponge - filter paper - filter paper - glue -PVDF membrane - filter paper - filter paper - sponge - white edge of clip";

4. The protein was electrotransferred to PVDF membrane. ( 0.45 µm PVDF membrane is recommended ) Reference Table for Selecting PVDF Membrane Pore Size Specifications

Recommended conditions for wet transfer: 200 mA, 60 min.

( Note that the transfer conditions can be adjusted according to the protein size. For high-molecular-weight proteins, a higher current and longer transfer time are recommended. However, ensure that the transfer tank remains at a low temperature to prevent gel melting.)

Block

1. After electrotransfer, wash the film with TBST at room temperature for 5 minutes;

2. Incubate the film in the blocking solution for 1 hour at room temperature;

3. Wash the film with TBST for 3 times, 5 minutes each time.

Antibody incubation

1. Use 5% skim milk powder to prepare the primary antibody working liquid (recommended dilution ratio for primary antibody 1:1000), gently shake and incubate with the film at 4°C overnight;

2. Wash the film with TBST 3 times, 5 minutes each time;

3. Add the secondary antibody to the blocking solution and incubate with the film gently at room temperature for 1 hour;

4. After incubation, wash the film with TBST 3 times for 5 minutes each time.

Antibody staining

1. Add the prepared ECL luminescent substrate (or select other color developing substrate according to the second antibody) and mix evenly;

2. Incubate with the film for 1 minute, remove excess substrate (keep the film moist), wrap with plastic film, and expose in the imaging system.

Datasheet & SDS

Biological Description

Specificity
BDNF Antibody (Rabbit mAb) [A7P4] detects endogenous levels of total BDNF protein.

Subcellular Location
Secreted

Uniprot ID
P23560

Clone
A7P4

Synonym(s)
Abrineurin; ANON2; BDNF; BDNF precursor form; brain derived neurotrophic factor; Brain-derived neurotrophic factor; BULN2; MGC34632; neurotrophin; ProBDNF

Background
BDNF (brain‑derived neurotrophic factor) is a dimeric secreted neurotrophin of the NGF/NT‑3/NT‑4 family that regulates neuronal growth, differentiation, survival, and synaptic plasticity across development and adulthood, and also modulates energy balance, behavior, and stress responses through actions in hypothalamic and limbic circuits. The precursor proBDNF is synthesized as a pre‑pro‑protein that is cleaved to proBDNF and further processed to mature BDNF (mBDNF); proBDNF preferentially engages the p75 neurotrophin receptor (p75NTR) often in complex with sortilin, while mBDNF binds with high affinity to the tropomyosin‑related kinase B receptor (TrkB), and these ligand–receptor pairings drive largely opposing synaptic outcomes. TrkB activation requires mBDNF dimerization and receptor dimerization with trans‑autophosphorylation of intracellular tyrosine residues that recruit Shc, Shp2, and PLCγ, initiating three major cascades: Ras–Raf–MEK–ERK signaling that supports neuronal differentiation, neurite growth, and activity‑dependent gene expression; PI3K–Akt–mTOR signaling that promotes survival, growth, and protein synthesis; and PLCγ–IP₃–DAG–PKC signaling with Ca²⁺ mobilization that underlies rapid modulation of synaptic strength. These pathways converge on transcription factors such as CREB and MEF2 and on local translation machinery, enabling BDNF to increase dendritic spine density, regulate AMPA and NMDA receptor trafficking, and stabilize long‑term potentiation in hippocampus and cortex, while proBDNF–p75NTR signaling enhances JNK activation, promotes long‑term depression, spine retraction, and in some contexts apoptotic or pruning‑like responses, establishing a bidirectional control system in which the balance of proBDNF and mBDNF shapes synaptic plasticity polarity. BDNF and TrkB are widely expressed throughout central and peripheral nervous systems, with high levels in hippocampus, cortex, amygdala, striatum, and hypothalamus; BDNF functions as a neurotransmitter modulator at excitatory and inhibitory synapses, regulates neurogenesis in adult hippocampal niches, and supports maturation and maintenance of multiple neuronal subtypes, including dopaminergic, serotonergic, and sensory neurons, while also influencing pain circuitry and stress axis regulation. Activity‑dependent transcription of BDNF is driven by Ca²⁺ influx through NMDA receptors and voltage‑gated Ca²⁺ channels, activation of CaMKs, ERK, and CREB, and promoter‑specific regulatory factors such as CaRF, allowing synaptic activity to induce BDNF expression and feed back onto synapses to implement synapse‑to‑nucleus‑to‑synapse signaling loops that underlie learning and memory. Circulating and brain BDNF levels are altered in numerous neurological and psychiatric disorders, including Alzheimer’s and Huntington’s diseases, major depression, bipolar disorder, schizophrenia, and anxiety, and BDNF Val66Met and other polymorphisms affect activity‑dependent secretion and have been associated with cognitive and affective phenotypes, supporting BDNF as both a mechanistic factor and a potential biomarker in CNS disease. BDNF expression in hypothalamic and mesolimbic circuits involved in feeding and reward links it to body‑weight regulation and energy metabolism, where BDNF–TrkB signaling influences satiety, locomotor activity, and fat oxidation via AMPK/ACC modulation, consistent with its requirement for normal appetite and energy expenditure.

Background

BDNF (brain‑derived neurotrophic factor) is a dimeric secreted neurotrophin of the NGF/NT‑3/NT‑4 family that regulates neuronal growth, differentiation, survival, and synaptic plasticity across development and adulthood, and also modulates energy balance, behavior, and stress responses through actions in hypothalamic and limbic circuits. The precursor proBDNF is synthesized as a pre‑pro‑protein that is cleaved to proBDNF and further processed to mature BDNF (mBDNF); proBDNF preferentially engages the p75 neurotrophin receptor (p75NTR) often in complex with sortilin, while mBDNF binds with high affinity to the tropomyosin‑related kinase B receptor (TrkB), and these ligand–receptor pairings drive largely opposing synaptic outcomes. TrkB activation requires mBDNF dimerization and receptor dimerization with trans‑autophosphorylation of intracellular tyrosine residues that recruit Shc, Shp2, and PLCγ, initiating three major cascades: Ras–Raf–MEK–ERK signaling that supports neuronal differentiation, neurite growth, and activity‑dependent gene expression; PI3K–Akt–mTOR signaling that promotes survival, growth, and protein synthesis; and PLCγ–IP₃–DAG–PKC signaling with Ca²⁺ mobilization that underlies rapid modulation of synaptic strength. These pathways converge on transcription factors such as CREB and MEF2 and on local translation machinery, enabling BDNF to increase dendritic spine density, regulate AMPA and NMDA receptor trafficking, and stabilize long‑term potentiation in hippocampus and cortex, while proBDNF–p75NTR signaling enhances JNK activation, promotes long‑term depression, spine retraction, and in some contexts apoptotic or pruning‑like responses, establishing a bidirectional control system in which the balance of proBDNF and mBDNF shapes synaptic plasticity polarity. BDNF and TrkB are widely expressed throughout central and peripheral nervous systems, with high levels in hippocampus, cortex, amygdala, striatum, and hypothalamus; BDNF functions as a neurotransmitter modulator at excitatory and inhibitory synapses, regulates neurogenesis in adult hippocampal niches, and supports maturation and maintenance of multiple neuronal subtypes, including dopaminergic, serotonergic, and sensory neurons, while also influencing pain circuitry and stress axis regulation. Activity‑dependent transcription of BDNF is driven by Ca²⁺ influx through NMDA receptors and voltage‑gated Ca²⁺ channels, activation of CaMKs, ERK, and CREB, and promoter‑specific regulatory factors such as CaRF, allowing synaptic activity to induce BDNF expression and feed back onto synapses to implement synapse‑to‑nucleus‑to‑synapse signaling loops that underlie learning and memory. Circulating and brain BDNF levels are altered in numerous neurological and psychiatric disorders, including Alzheimer’s and Huntington’s diseases, major depression, bipolar disorder, schizophrenia, and anxiety, and BDNF Val66Met and other polymorphisms affect activity‑dependent secretion and have been associated with cognitive and affective phenotypes, supporting BDNF as both a mechanistic factor and a potential biomarker in CNS disease. BDNF expression in hypothalamic and mesolimbic circuits involved in feeding and reward links it to body‑weight regulation and energy metabolism, where BDNF–TrkB signaling influences satiety, locomotor activity, and fat oxidation via AMPK/ACC modulation, consistent with its requirement for normal appetite and energy expenditure.

References
https://pubmed.ncbi.nlm.nih.gov/34071978/ https://pubmed.ncbi.nlm.nih.gov/33096634/

Protein Size (kDa)	PVDF Transfer Membrane Pore Size (μm)
< 10	0.22
10-20	0.22
20-30	0.45
30-45	0.45
45-70	0.45
80-100	0.45
100-140	0.45
> 140	0.45

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Protein Size (kDa)	Separating Gel Percentage
4-40	20%
12-45	15%
10-70	12.5%
15-100	10%
25-100	8%
60-210	5%

BDNF Antibody (Rabbit mAb) [A7P4]