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생물학적 설명

특이성	Phospho-IRS-1 (Ser302) Antibody [K1C11]는 Ser 302에서 인산화된 경우에만 내인성 IRS-1 수준을 검출합니다.
배경	인슐린 수용체 기질 1 (IRS-1)의 세린/트레오닌 잔기 인산화는 인슐린 신호 조절에 중요한 역할을 합니다. IRS-1은 인슐린 수용체 키나제의 주요 기질이며, SH2 도메인을 포함하는 단백질의 도킹 플랫폼 역할을 하는 다수의 티로신 인산화 부위를 포함하여 인슐린의 대사 및 성장 촉진 작용을 촉진합니다. IRS 단백질은 다수의 상호작용 도메인 및 인산화 모티프를 특징으로 하지만, 내재적 촉매 활성이 부족합니다. 각 IRS 단백질은 NH2-말단 플렉스트린 상동성 도메인과 인산화 티로신 결합 도메인을 특징으로 하며, 이는 IRS 단백질이 인슐린, IGF-I, IL-4와 같은 활성화된 수용체에 연결되도록 돕습니다. 인슐린은 근육 및 지방 세포에서 영양소 흡수 및 저장을 촉진하고 간에서 포도당 신생합성을 억제하므로 대사 조절에 중심적인 역할을 합니다. 사이토카인 유도 세린 인산화, IRS 단백질 분해 또는 인슐린 수용체의 직접적인 억제를 포함한 여러 메커니즘이 인슐린 신호 전달 경로를 조절할 수 있습니다. IRS-1은 다양한 키나제에 의해 표적화될 수 있는 20개 이상의 잠재적인 세린 인산화 부위를 가지고 있습니다. 예를 들어, Ser302의 인산화는 인슐린 유도 IRS-1의 티로신 인산화에 필요하며, 이는 인슐린 신호와 영양소 가용성을 잠재적으로 연결합니다. 한편, PKCθ에 의해 매개되는 Ser1101에서 IRS-1의 인산화는 인슐린 신호를 억제하여 일부 비만 모델에서 관찰되는 인슐린 저항성에 대한 잠재적인 설명을 제공합니다.

특이성

Phospho-IRS-1 (Ser302) Antibody [K1C11]는 Ser 302에서 인산화된 경우에만 내인성 IRS-1 수준을 검출합니다.

배경

인슐린 수용체 기질 1 (IRS-1)의 세린/트레오닌 잔기 인산화는 인슐린 신호 조절에 중요한 역할을 합니다. IRS-1은 인슐린 수용체 키나제의 주요 기질이며, SH2 도메인을 포함하는 단백질의 도킹 플랫폼 역할을 하는 다수의 티로신 인산화 부위를 포함하여 인슐린의 대사 및 성장 촉진 작용을 촉진합니다. IRS 단백질은 다수의 상호작용 도메인 및 인산화 모티프를 특징으로 하지만, 내재적 촉매 활성이 부족합니다. 각 IRS 단백질은 NH2-말단 플렉스트린 상동성 도메인과 인산화 티로신 결합 도메인을 특징으로 하며, 이는 IRS 단백질이 인슐린, IGF-I, IL-4와 같은 활성화된 수용체에 연결되도록 돕습니다. 인슐린은 근육 및 지방 세포에서 영양소 흡수 및 저장을 촉진하고 간에서 포도당 신생합성을 억제하므로 대사 조절에 중심적인 역할을 합니다. 사이토카인 유도 세린 인산화, IRS 단백질 분해 또는 인슐린 수용체의 직접적인 억제를 포함한 여러 메커니즘이 인슐린 신호 전달 경로를 조절할 수 있습니다. IRS-1은 다양한 키나제에 의해 표적화될 수 있는 20개 이상의 잠재적인 세린 인산화 부위를 가지고 있습니다. 예를 들어, Ser302의 인산화는 인슐린 유도 IRS-1의 티로신 인산화에 필요하며, 이는 인슐린 신호와 영양소 가용성을 잠재적으로 연결합니다. 한편, PKCθ에 의해 매개되는 Ser1101에서 IRS-1의 인산화는 인슐린 신호를 억제하여 일부 비만 모델에서 관찰되는 인슐린 저항성에 대한 잠재적인 설명을 제공합니다.

사용 정보

응용

WB

희석

WB

1:1000

반응성

Human, Mouse

출처

Rabbit Monoclonal Antibody

MW

180 kDa

보관 완충액

PBS, pH 7.2+50% Glycerol+0.05% BSA+0.01% NaN₃

보관
(수령일로부터)

-20°C (avoid freeze-thaw cycles), 2 years

실험 방법
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: 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 1117. 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. (Exposure time of at least 90s is recommended)

실험 방법

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: 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

1117. 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. (Exposure time of at least 90s is recommended)

참조

https://pubmed.ncbi.nlm.nih.gov/14623899/

적용 데이터

WB

Selleck 검증

Lane 1: MCF7 (Insulin, 100 nM, 5 min)
Lane 2: C2C12 (Insulin, 100 nM, 5 min)