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Anti-Phospho-IGF-1R β (Y1131)/IR β (Y1146) Rabbit Antibody [J6K20]

Catalog No.: F3033

Application: Reactivity: Human

Usage Information

Dilution
WB1:1000 IP1:100

Application
WB, IP

Reactivity
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
95 kDa

Positive Control	293 (serum-starved overnight, IGF-I, 100 nM, 2 min)
Negative Control	293 (serum-starved overnight)

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: 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 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: 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 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-IGF-I Receptor β (Tyr1131)/Insulin Receptor β (Tyr1146) Rabbit mAb detects endogenous levels of IGF-I receptor β protein only when phosphorylated at Tyr1131 and/or insulin receptor β protein only when phosphorylated at Tyr1146. The antibody cross-reacts with activated ErbB2 and ALK.

Subcellular Location
Cell membrane, Membrane

Uniprot ID
P08069

Clone
J6K20

Synonym(s)
Insulin-like growth factor 1 receptor, Insulin-like growth factor I receptor (IGF-I receptor), CD221, IGF1R

Background
The vascular insulin-like growth factor-1 (IGF-1) system is composed of IGFs, the IGF-1 receptor (IGF-1R), and a range of IGF-binding proteins. Acting through both endocrine and autocrine/paracrine routes, this growth factor network influences numerous vascular functions. Although IGF-1 activity is primarily mediated by IGF-1R, it is finely tuned by the IGF-binding proteins, whose functions are regulated through phosphorylation, proteolytic cleavage, polymerization, and association with cells or the extracellular matrix. The expression of IGF system components is controlled by diverse stimuli, including other growth factors, cytokines, lipoproteins, reactive oxygen species, and hemodynamic forces. Furthermore, cross-communication has been observed between the IGF system and various growth factor pathways as well as integrin receptors. In humans, IGF-1R is encoded by a single-copy gene on chromosome 15 and is expressed ubiquitously. The mature receptor is a tetramer, composed of two extracellular α-chains and two intracellular β-chains. The β-chains harbor an intracellular tyrosine kinase domain, which is critical for most receptor-mediated biological functions. Upon IGF-1 binding, IGF-1R undergoes autophosphorylation, followed by tyrosine phosphorylation of adaptor proteins such as Shc and insulin receptor substrates (IRS-1, IRS-2, IRS-3, and IRS-4). IRS proteins function as docking platforms that trigger several downstream signaling cascades, notably phosphatidylinositol 3-kinase (PI3K), Akt, and mitogen-activated protein kinase (MAPK) pathways. These signaling events lead to distinct cellular responses, including proliferation, differentiation, migration, and survival. The initial autophosphorylation within the IGF-1R kinase domain takes place at three key tyrosine residues—Tyr1131, Tyr1135, and Tyr1136. Insulin receptors (IRs) exhibit strong structural and functional resemblance to IGF-1R, possessing an analogous tyrosine cluster—Tyr1146, Tyr1150, and Tyr1151—positioned within the activation loop of their kinase domain.

Background

The vascular insulin-like growth factor-1 (IGF-1) system is composed of IGFs, the IGF-1 receptor (IGF-1R), and a range of IGF-binding proteins. Acting through both endocrine and autocrine/paracrine routes, this growth factor network influences numerous vascular functions. Although IGF-1 activity is primarily mediated by IGF-1R, it is finely tuned by the IGF-binding proteins, whose functions are regulated through phosphorylation, proteolytic cleavage, polymerization, and association with cells or the extracellular matrix. The expression of IGF system components is controlled by diverse stimuli, including other growth factors, cytokines, lipoproteins, reactive oxygen species, and hemodynamic forces. Furthermore, cross-communication has been observed between the IGF system and various growth factor pathways as well as integrin receptors. In humans, IGF-1R is encoded by a single-copy gene on chromosome 15 and is expressed ubiquitously. The mature receptor is a tetramer, composed of two extracellular α-chains and two intracellular β-chains. The β-chains harbor an intracellular tyrosine kinase domain, which is critical for most receptor-mediated biological functions. Upon IGF-1 binding, IGF-1R undergoes autophosphorylation, followed by tyrosine phosphorylation of adaptor proteins such as Shc and insulin receptor substrates (IRS-1, IRS-2, IRS-3, and IRS-4). IRS proteins function as docking platforms that trigger several downstream signaling cascades, notably phosphatidylinositol 3-kinase (PI3K), Akt, and mitogen-activated protein kinase (MAPK) pathways. These signaling events lead to distinct cellular responses, including proliferation, differentiation, migration, and survival. The initial autophosphorylation within the IGF-1R kinase domain takes place at three key tyrosine residues—Tyr1131, Tyr1135, and Tyr1136. Insulin receptors (IRs) exhibit strong structural and functional resemblance to IGF-1R, possessing an analogous tyrosine cluster—Tyr1146, Tyr1150, and Tyr1151—positioned within the activation loop of their kinase domain.

References
https://pubmed.ncbi.nlm.nih.gov/14604834/ https://pubmed.ncbi.nlm.nih.gov/3848433/

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
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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%