Phospho-Insulin Receptor β (Tyr1345) Antibody [L11H12]

Application: Reactivity: Human

Lane 1: CHO (INSR(human)-EGFP transfected), Lane 2: CHO (INSR(human)-EGFP transfected; insulin, 100 nM, 5 min)

Usage Information

Dilution
WB1:1000

Application
WB

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
95 kDa

Positive Control	CHO IR/IRS-1 cells (Insulin, 100 nM, 5 min)
Negative Control	CHO IR/IRS-1 cells

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, Phosphatase 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, Phosphatase 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, Phosphatase 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, 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 primary antibody dilution buffer 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, Phosphatase 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, Phosphatase 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, Phosphatase 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, 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 primary antibody dilution buffer 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-Insulin Receptor β (Tyr1345) Antibody (Rabbit mAb) [L11H12] detects endogenous levels of total Insulin Receptor β protein only when it is phosphorylated at Tyr1345.

Subcellular Location
Cell membrane, Endosome, Lysosome, Membrane

Uniprot ID
P06213

Clone
L11H12

Synonym(s)
Insulin receptor, IR, Insulin receptor subunit beta, CD220, INSR

Background
Phospho‑insulin receptor β (Tyr1345) represents an activated state of the β‑subunit of the insulin receptor, a receptor tyrosine kinase of the insulin/IGF receptor family that controls glucose uptake, metabolism, and growth signaling through ligand‑induced autophosphorylation and recruitment of downstream effectors. The receptor is synthesized as a single precursor that is cleaved into extracellular α and transmembrane β chains, with the β‑subunit carrying the intracellular kinase domain, an activation loop tyrosine cluster that governs catalytic activity, and multiple additional tyrosines including Tyr1345 that serve as regulatory and docking sites once phosphorylated. Autophosphorylation is initiated when insulin binding to the extracellular α‑subunits brings two receptor dimers together, allowing trans‑phosphorylation of the activation loop tyrosines in the kinase domain, which relieves autoinhibition and increases kinase activity toward both receptor tyrosines and cytoplasmic substrates such as IRS proteins and SHC. Subsequent phosphorylation at more distal sites in the C‑terminal tail, including Tyr1345, expands the repertoire of SH2‑domain–containing partners that recognize the receptor, supporting assembly of multi‑protein complexes involved in fine‑tuning signaling strength, receptor endocytosis, and lysosomal targeting. Once activated, phospho‑insulin receptor β drives phosphorylation of IRS1–4, which then recruit PI3K p85 to initiate PI3K–AKT signaling, leading to PIP3 generation, activation of PDK1 and AKT, and downstream events such as GLUT4 translocation to the plasma membrane, suppression of gluconeogenic gene expression through FOXO phosphorylation, and stimulation of mTORC1‑dependent protein synthesis via TSC2 phosphorylation. Parallel phosphorylation of SHC and other adaptors promotes GRB2–SOS recruitment and activation of the RAS–RAF–MEK–ERK pathway, which contributes to gene expression programs linked to growth, survival, and differentiation and cooperates with PI3K–AKT outputs to coordinate metabolic and mitogenic responses to insulin. Phospho‑Tyr1345 is associated with receptor internalization to endosomal and lysosomal compartments, providing a handle to monitor receptor trafficking and down‑regulation, and antibodies directed to this site selectively detect the activated, phosphorylated receptor β‑chain against a background of constant total receptor levels. Dysregulated insulin receptor phosphorylation patterns, including altered kinetics or amplitude of distal β‑subunit phosphotyrosines, accompany states of insulin resistance and type 2 diabetes, where imbalanced activation and termination of PI3K–AKT versus RAS–MAPK signaling contribute to impaired glucose handling and maladaptive growth and survival signaling in metabolic tissues.

Background

Phospho‑insulin receptor β (Tyr1345) represents an activated state of the β‑subunit of the insulin receptor, a receptor tyrosine kinase of the insulin/IGF receptor family that controls glucose uptake, metabolism, and growth signaling through ligand‑induced autophosphorylation and recruitment of downstream effectors. The receptor is synthesized as a single precursor that is cleaved into extracellular α and transmembrane β chains, with the β‑subunit carrying the intracellular kinase domain, an activation loop tyrosine cluster that governs catalytic activity, and multiple additional tyrosines including Tyr1345 that serve as regulatory and docking sites once phosphorylated. Autophosphorylation is initiated when insulin binding to the extracellular α‑subunits brings two receptor dimers together, allowing trans‑phosphorylation of the activation loop tyrosines in the kinase domain, which relieves autoinhibition and increases kinase activity toward both receptor tyrosines and cytoplasmic substrates such as IRS proteins and SHC. Subsequent phosphorylation at more distal sites in the C‑terminal tail, including Tyr1345, expands the repertoire of SH2‑domain–containing partners that recognize the receptor, supporting assembly of multi‑protein complexes involved in fine‑tuning signaling strength, receptor endocytosis, and lysosomal targeting. Once activated, phospho‑insulin receptor β drives phosphorylation of IRS1–4, which then recruit PI3K p85 to initiate PI3K–AKT signaling, leading to PIP3 generation, activation of PDK1 and AKT, and downstream events such as GLUT4 translocation to the plasma membrane, suppression of gluconeogenic gene expression through FOXO phosphorylation, and stimulation of mTORC1‑dependent protein synthesis via TSC2 phosphorylation. Parallel phosphorylation of SHC and other adaptors promotes GRB2–SOS recruitment and activation of the RAS–RAF–MEK–ERK pathway, which contributes to gene expression programs linked to growth, survival, and differentiation and cooperates with PI3K–AKT outputs to coordinate metabolic and mitogenic responses to insulin. Phospho‑Tyr1345 is associated with receptor internalization to endosomal and lysosomal compartments, providing a handle to monitor receptor trafficking and down‑regulation, and antibodies directed to this site selectively detect the activated, phosphorylated receptor β‑chain against a background of constant total receptor levels. Dysregulated insulin receptor phosphorylation patterns, including altered kinetics or amplitude of distal β‑subunit phosphotyrosines, accompany states of insulin resistance and type 2 diabetes, where imbalanced activation and termination of PI3K–AKT versus RAS–MAPK signaling contribute to impaired glucose handling and maladaptive growth and survival signaling in metabolic tissues.

References
https://pubmed.ncbi.nlm.nih.gov/1321605/ https://pubmed.ncbi.nlm.nih.gov/27512793/

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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Handling Instructions

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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%

Phospho-Insulin Receptor β (Tyr1345) Antibody (Rabbit mAb) [L11H12]

Usage Information

Experimental Methods

Datasheet & SDS

Biological Description

Tech Support