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HER3/ErbB3 Antibody [N12M8]

Cat.No.: F4189

Application: Reactivity: Human

Lane 1: T47D, Lane 2: MCF-7

Experiment Essentials

WB
Recommended SDS-PAGE separating gel concentration: 5%.

Usage Information

Dilution
WB1:1000 IP1:50

Application
WB, IP

Reactivity
Human

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
185 kDa

Positive Control	T47D cell; MCF7 cell
Negative Control

Exprimental 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. 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) 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 RIPA/NP-40 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: 5%. 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 RIPA/NP-40 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 RIPA/NP-40 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 RIPA/NP-40 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: 5%. 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
HER3/ErbB3 Antibody [N12M8] detects endogenous levels of total HER3/ErbB3 protein.

Subcellular Location
Cell membrane, Membrane, Secreted

Uniprot ID
P21860

Clone
N12M8

Synonym(s)
Receptor tyrosine-protein kinase erbB-3; Proto-oncogene-like protein c-ErbB-3; Tyrosine kinase-type cell surface receptor HER3; ERBB3; HER3

Background
HER3/ErbB3 is a catalytically inactive “pseudokinase” member of the ErbB/HER receptor tyrosine kinase family, rendered intrinsically inactive by key substitutions in its kinase domain but capable of robust signaling through heterodimerization with active family members such as HER2 or EGFR. HER3 comprises an extracellular domain (ECD) with four subdomains: leucine-rich I/III for high-affinity neuregulin (NRG1/2) binding and cysteine-rich II/IV, with subdomain II harboring a dimerization arm; a single transmembrane α-helix; a juxtamembrane region; an atypical kinase domain (shortened αC-helix, inactive configuration despite ATP binding); and a C-terminal tail containing at least 9–11 tyrosine sites (notably Tyr1222 and Tyr1289 in YXXM motifs) for adaptor docking. Upon ligand binding, HER3’s ECD undergoes a conformational change that exposes the dimerization loop, promoting asymmetric heterodimer formation, most potently with HER2, whereby the partner’s kinase activity trans-phosphorylates HER3’s tyrosines. These phosphorylated tyrosines recruit PI3K p85 (via YXXM motifs), Grb2/Shc (MAPK/ERK), and Src, activating pathways for cell survival and proliferation. HER3 is crucial for organogenesis (nervous and cardiac development), drives cancer progression notably in breast, ovarian, and lung cancers, and mediates resistance to EGFR/HER2 inhibitors through compensatory signaling.

Background

HER3/ErbB3 is a catalytically inactive “pseudokinase” member of the ErbB/HER receptor tyrosine kinase family, rendered intrinsically inactive by key substitutions in its kinase domain but capable of robust signaling through heterodimerization with active family members such as HER2 or EGFR. HER3 comprises an extracellular domain (ECD) with four subdomains: leucine-rich I/III for high-affinity neuregulin (NRG1/2) binding and cysteine-rich II/IV, with subdomain II harboring a dimerization arm; a single transmembrane α-helix; a juxtamembrane region; an atypical kinase domain (shortened αC-helix, inactive configuration despite ATP binding); and a C-terminal tail containing at least 9–11 tyrosine sites (notably Tyr1222 and Tyr1289 in YXXM motifs) for adaptor docking. Upon ligand binding, HER3’s ECD undergoes a conformational change that exposes the dimerization loop, promoting asymmetric heterodimer formation, most potently with HER2, whereby the partner’s kinase activity trans-phosphorylates HER3’s tyrosines. These phosphorylated tyrosines recruit PI3K p85 (via YXXM motifs), Grb2/Shc (MAPK/ERK), and Src, activating pathways for cell survival and proliferation. HER3 is crucial for organogenesis (nervous and cardiac development), drives cancer progression notably in breast, ovarian, and lung cancers, and mediates resistance to EGFR/HER2 inhibitors through compensatory signaling.

References
https://pubmed.ncbi.nlm.nih.gov/20351256/ https://pubmed.ncbi.nlm.nih.gov/20007378/

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

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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%