Phospho-IGF-1R (Y1135/1136)/IR (Y1150/1151) Antibody [B2J21]

Catalog No.: F0247

Application: Reactivity: Human, Mouse, Rat

Lane 1: Hela, Lane 2: Hela (serum starvation and IGF-treated), Lane 3: H-4-II-E, Lane 4: H-4-II-E (serum starvation and insulin-treated)
Immunohistochemical analysis of formalin fixed paraffin embedded human Colorectal cancer tissue with F0247 at 1/100 dilution.

サイズ（液体）	価格(税別)	在庫状況
20uL	JPY 30400	国内在庫あり
100uL	JPY 67200	国内在庫あり
100uL*2	JPY 99900	お問い合わせ

代表番号: 045-509-1970\|電子メール：sales@selleck.co.jp よく尋ねられる質問

カスタマーフィードバック(1)

キーポイント

WB
Phospho-IGF-I Receptor (Tyr 1135/1136)の基底発現は低いため、事前に誘導刺激を行うことを推奨します。

使用情報

Dilution
WB1:1000

Application
WB

Source
Rabbit Monoclonal Antibody

Reactivity
Human, Mouse, Rat

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

ポジティブコントロール	Hela (IGF-treated); H-4-II-E (insulin-treated)
ネガティブコントロール	Hela; H-4-II-E

サンプル処理データの例

サンプル	処理状況
Hela	IGF
H-4-II-E	Insulin
クリックして、さらに多くのサンプルデータを表示

^*異なるヒト由来細胞や組織における発現量の予測については、以下をご参照ください: http://www.proteinatlas.org

サンプル	処理状況

プロトコール

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 transfer the cells into an EP tube. Wash the cells with ice-cold PBS twice. Add 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.Add 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 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 938. 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 or lyse it by sonication on ice, then incubate on ice for 30 minutes.

2. Adherent cell: Aspirate the culture medium and transfer the cells into an EP tube. Wash the cells with ice-cold PBS twice. Add 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.Add 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 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

938. 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

生物学的記述

Specificity
Phospho-IGF-1R (Y1135/1136)/IR (Y1150/1151) Antibody [B2J21] detects endogenous levels of IGF-I receptor and insulin receptor only when phosphorylated at Tyr1135/1136 or Tyr1150/1151, respectively. It does not cross-react with other related tyrosine-phosphorylated tyrosine kinases.

タンパク質の局在
細胞膜、細胞内膜系、エンドソーム、リソソーム

Uniprot ID
P08069, P06213

Clone
B2J21

Synonym(s)
Phospho IGFIR (Tyr1135/1136), Phospho IR (Tyr1150/1151)

Background
Phospho-IGF-I Receptor β (Tyr1135/1136) and Insulin Receptor β (Tyr1150/1151) are phosphorylated forms of specific tyrosine residues on the β subunits of the IGF-1 receptor (IGF-1R) and insulin receptor (IR), respectively. These receptors are heterotetrameric transmembrane tyrosine kinases composed of two alpha and two beta subunits. Upon ligand binding, such as insulin or IGF-1, the receptors undergo autophosphorylation at key tyrosine residues, including Tyr1135/1136 on IGF-1R and Tyr1150/1151 on IR, which are part of a critical tyrosine cluster within the kinase domain. This autophosphorylation is essential for receptor activation, enabling them to phosphorylate downstream signaling molecules, thereby initiating various intracellular signaling cascades involved in cellular growth, metabolism, and survival. Phosphorylation of these tyrosines is necessary for the full activation of the receptor’s kinase activity and subsequent cellular responses to insulin and IGF-1.

Background

Phospho-IGF-I Receptor β (Tyr1135/1136) and Insulin Receptor β (Tyr1150/1151) are phosphorylated forms of specific tyrosine residues on the β subunits of the IGF-1 receptor (IGF-1R) and insulin receptor (IR), respectively. These receptors are heterotetrameric transmembrane tyrosine kinases composed of two alpha and two beta subunits. Upon ligand binding, such as insulin or IGF-1, the receptors undergo autophosphorylation at key tyrosine residues, including Tyr1135/1136 on IGF-1R and Tyr1150/1151 on IR, which are part of a critical tyrosine cluster within the kinase domain. This autophosphorylation is essential for receptor activation, enabling them to phosphorylate downstream signaling molecules, thereby initiating various intracellular signaling cascades involved in cellular growth, metabolism, and survival. Phosphorylation of these tyrosines is necessary for the full activation of the receptor’s kinase activity and subsequent cellular responses to insulin and IGF-1.

References
[1] Hernández-Sánchez C, et al. J Biol Chem. 1995 Dec 8;270(49):29176-81. [2] White MF, et al. J Biol Chem. 1988 Feb 25;263(6):2969-80.

PVDFメンブレン孔径参考表

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%