Choose Your Country or Region

Anti-Netrin 1 Rabbit Antibody [B4C16]

Catalog No.: F1639

Application: Reactivity: Mouse, Rat, Human

Experiment Essentials

WB
We recommend using 1% SDS heat lysis for sample preparation. Conventional methods may result in weak protein signals.

Usage Information

Dilution
WB1:500 - 1:10000 IP1:1500

Application
WB, IP

Reactivity
Mouse, Rat, Human

Source
Rabbit

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
67 kDa 75 kDa
^*Why do the predicted and actual molecular weights differ? The following reasons may explain differences between the predicted and actual protein molecular weight.

Positive Control	Rat brain (prepared in 1%SDS Hot lysis method at 20 µg); Mouse kidney tissue; Mouse heart tissue; Rat kidney tissue; Human fetal brain tissue; Human small intestine; Mouse aorta (Ang II-treated); MCF7
Negative Control	Mouse aorta (PBS-treated); Rat brain (prepared in RIPA lysis method)

Exprimental Methods

WB
Experimental Protocol: Sample preparation 1. Tissue: Lyse the tissue sample by adding an appropriate volume of ice-cold Hot 1% SDS Lysis Buffer (containing Protease Inhibitor Cocktail)，and homogenize the tissue at a low temperature. 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 Hot 1% SDS Lysis Buffer (containing Protease Inhibitor Cocktail) and put the sample on ice for 5 min. 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 Hot 1% SDS Lysis Buffer (containing Protease Inhibitor Cocktail) and put the sample on ice for 5 min. 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, 120 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.

Experimental Protocol:

Sample preparation

1. Tissue: Lyse the tissue sample by adding an appropriate volume of ice-cold Hot 1% SDS Lysis Buffer (containing Protease Inhibitor Cocktail)，and homogenize the tissue at a low temperature.

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 Hot 1% SDS Lysis Buffer (containing Protease Inhibitor Cocktail) and put the sample on ice for 5 min.

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 Hot 1% SDS Lysis Buffer (containing Protease Inhibitor Cocktail) and put the sample on ice for 5 min.

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, 120 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
Anti-Netrin 1 Rabbit Antibody [B4C16] detects endogenous levels of total Netrin 1 protein.

Subcellular Location
Cytoplasm, Secreted

Uniprot ID
O95631

Clone
B4C16

Synonym(s)
NTN1L, NTN1, Netrin-1, Epididymis tissue protein Li 131P

Background
Netrin-1 is a multifunctional guidance cue protein belonging to the netrin family, primarily recognized for its critical role in nervous system development by directing axonal growth, neuronal migration, and synapse formation. It is composed of an N-terminal laminin-like domain followed by three epidermal growth factor (EGF)-like repeats and a small positively charged C-terminal domain. This unique structure allows netrin-1 to interact with multiple receptors simultaneously, such as DCC and UNC5, enabling it to trigger either attractive or repulsive signaling pathways. These pathways steer growing axons to their targets by modulating cytoskeletal dynamics and cell adhesion machinery within the growth cone, a dynamic structure at the axon's tip. Netrin-1 regulates angiogenesis by promoting endothelial cell survival, migration, and blood vessel formation, and modulates immune cell trafficking by influencing leukocyte adhesion and migration. It also mediates cell survival through inhibition of apoptosis by activating pro-survival pathways and suppressing caspase activity. Netrin-1 signaling is tightly controlled spatially and temporally, with expression regulated by developmental cues and environmental factors. Its interaction with multiple co-receptors and extracellular matrix components allows versatile cellular responses depending on context. Netrin-1 is implicated in cancer progression by fostering tumor cell survival, enhancing metastatic spread, and modulating the tumor microenvironment through angiogenesis and immune evasion.

Background

Netrin-1 is a multifunctional guidance cue protein belonging to the netrin family, primarily recognized for its critical role in nervous system development by directing axonal growth, neuronal migration, and synapse formation. It is composed of an N-terminal laminin-like domain followed by three epidermal growth factor (EGF)-like repeats and a small positively charged C-terminal domain. This unique structure allows netrin-1 to interact with multiple receptors simultaneously, such as DCC and UNC5, enabling it to trigger either attractive or repulsive signaling pathways. These pathways steer growing axons to their targets by modulating cytoskeletal dynamics and cell adhesion machinery within the growth cone, a dynamic structure at the axon's tip. Netrin-1 regulates angiogenesis by promoting endothelial cell survival, migration, and blood vessel formation, and modulates immune cell trafficking by influencing leukocyte adhesion and migration. It also mediates cell survival through inhibition of apoptosis by activating pro-survival pathways and suppressing caspase activity. Netrin-1 signaling is tightly controlled spatially and temporally, with expression regulated by developmental cues and environmental factors. Its interaction with multiple co-receptors and extracellular matrix components allows versatile cellular responses depending on context. Netrin-1 is implicated in cancer progression by fostering tumor cell survival, enhancing metastatic spread, and modulating the tumor microenvironment through angiogenesis and immune evasion.

References
https://pubmed.ncbi.nlm.nih.gov/24876346/ https://pubmed.ncbi.nlm.nih.gov/25123307/

Reference Table for Selecting PVDF Membrane Pore Size Specifications

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

Tech Support

Answers to questions you may have can be found in the inhibitor handling instructions. Topics include how to prepare stock solutions, how to store inhibitors, and issues that need special attention for cell-based assays and animal experiments.

Handling Instructions

Tel: +1-832-582-8158 Ext:3
If you have any other enquiries, please leave a message.

* Indicates a Required Field

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%