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Phospho-HER2/ErbB2 (Tyr1196) Antibody [L1F9]

Cat.No.: F0853

Application: Reactivity: Human, Mouse

Lane 1: MCF7, Lane 2: MCF7 (human neuregulin-1, 100 ng/ml, 5 min)

Experiment Essentials

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

Usage Information

Dilution
WB1:1000 IP1:100

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
185 kDa

Positive Control	T47D (human neuregulin-1, 100 ng/ml, 5 min); MCF7 (human neuregulin-1, 100 ng/ml, 5 min)
Negative Control	T47D; MCF7

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, Phosphatase 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, Phosphatase 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, Phosphatase 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 ( recommending 5% BSA 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, Phosphatase 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, Phosphatase 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, Phosphatase 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 ( recommending 5% BSA 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
Phospho-HER2/ErbB2 (Tyr1196) Antibody [L1F9] recognizes endogenous levels of HER2/ErbB2 protein only when phosphorylated at Tyr1196. The antibody may detect other activated ErbB family members.

Subcellular Location
Cell membrane, Cell projection, Cytoplasm, Endosome, Membrane,Nucleus

Uniprot ID
P04626

Clone
L1F9

Synonym(s)
Receptor tyrosine-protein kinase erbB-2, Metastatic lymph node gene 19 protein (MLN 19), Proto-oncogene Neu, Proto-oncogene c-ErbB-2, Tyrosine kinase-type cell surface receptor HER2, p185erbB2, ERBB2, HER2, MLN19, NEU, NGL

Background
The ErbB2 proto-oncogene, also known as HER2, encodes a 185 kDa transmembrane glycoprotein that functions as a receptor with intrinsic tyrosine kinase activity. Although ErbB2 does not have a known ligand, its kinase activity can be triggered independently of ligand binding, particularly when overexpressed or through dimerization with other members of the ErbB receptor family. ErbB2 gene amplification and protein overexpression occur in approximately 40% of human breast cancers, making it a critical therapeutic target for breast and other epithelial cancers. One potential approach to therapy involves enhancing the degradation of ErbB2 via a c-Cbl-regulated proteolytic pathway. Phosphorylation at tyrosine residue 877, located within the kinase domain and analogous to Tyr416 in pp60c-Src, may influence ErbB2's functional activity. Key autophosphorylation sites on ErbB2 include Tyr1248 and Tyr1221/1222, which facilitate signaling through the Ras-Raf-MAPK pathway. Additionally, autophosphorylation at Tyr1196 promotes interaction with the Crk adaptor protein, leading to Erk activation through a Ras-independent mechanism.

Background

The ErbB2 proto-oncogene, also known as HER2, encodes a 185 kDa transmembrane glycoprotein that functions as a receptor with intrinsic tyrosine kinase activity. Although ErbB2 does not have a known ligand, its kinase activity can be triggered independently of ligand binding, particularly when overexpressed or through dimerization with other members of the ErbB receptor family. ErbB2 gene amplification and protein overexpression occur in approximately 40% of human breast cancers, making it a critical therapeutic target for breast and other epithelial cancers. One potential approach to therapy involves enhancing the degradation of ErbB2 via a c-Cbl-regulated proteolytic pathway. Phosphorylation at tyrosine residue 877, located within the kinase domain and analogous to Tyr416 in pp60c-Src, may influence ErbB2's functional activity. Key autophosphorylation sites on ErbB2 include Tyr1248 and Tyr1221/1222, which facilitate signaling through the Ras-Raf-MAPK pathway. Additionally, autophosphorylation at Tyr1196 promotes interaction with the Crk adaptor protein, leading to Erk activation through a Ras-independent mechanism.

References
https://pubmed.ncbi.nlm.nih.gov/17274834/ https://pubmed.ncbi.nlm.nih.gov/11238891/

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%