데이터시트

생물학적 설명

특이성	Phospho-PDGF Receptor β (Tyr1021) Antibody [M20P20]는 티로신 1021에서 인산화된 경우에만 내인성 PDGF β 수용체 수준을 검출합니다. 이 항체는 다른 활성화된 PDGF 수용체 계열 구성원 및 EGFR을 포함한 다른 활성화된 Protein Tyrosine Kinase와 교차 반응할 수 있습니다.
배경	Phospho-PDGF Receptor β (Tyr 1021)은 수용체 활성화 및 신호 전달을 위한 중요한 사건인 티로신 1021에서 인산화된 혈소판 유래 성장 인자 수용체 베타(PDGFRβ)의 활성화된 형태를 나타냅니다. PDGFRβ는 5개의 면역글로불린 유사 영역을 가진 세포외 도메인, 단일 막관통 도메인 및 세포내 Protein Tyrosine Kinase 도메인으로 구성된 수용체 Protein Tyrosine Kinase입니다. 주로 혈관 평활근 세포, 섬유아세포, 내피 세포 및 주위 세포에서 발현되며, 섬유증 및 암과 같은 손상 또는 질병에 대한 반응으로 발현이 상향 조절됩니다. Tyr 1021에서의 인산화는 Grb2 및 PLC-γ와 같은 신호 분자를 모집하여 세포 증식, 이동, 생존 및 혈관신생을 조절하는 경로를 시작합니다. 이 인산화는 조직 복구 및 발달에서 PDGFRβ의 역할에 필수적이며, 그 조절 장애는 암 및 섬유증과 같은 병리학적 상태에 기여하므로 잠재적인 치료 표적이 됩니다.

특이성

Phospho-PDGF Receptor β (Tyr1021) Antibody [M20P20]는 티로신 1021에서 인산화된 경우에만 내인성 PDGF β 수용체 수준을 검출합니다. 이 항체는 다른 활성화된 PDGF 수용체 계열 구성원 및 EGFR을 포함한 다른 활성화된 Protein Tyrosine Kinase와 교차 반응할 수 있습니다.

배경

Phospho-PDGF Receptor β (Tyr 1021)은 수용체 활성화 및 신호 전달을 위한 중요한 사건인 티로신 1021에서 인산화된 혈소판 유래 성장 인자 수용체 베타(PDGFRβ)의 활성화된 형태를 나타냅니다. PDGFRβ는 5개의 면역글로불린 유사 영역을 가진 세포외 도메인, 단일 막관통 도메인 및 세포내 Protein Tyrosine Kinase 도메인으로 구성된 수용체 Protein Tyrosine Kinase입니다. 주로 혈관 평활근 세포, 섬유아세포, 내피 세포 및 주위 세포에서 발현되며, 섬유증 및 암과 같은 손상 또는 질병에 대한 반응으로 발현이 상향 조절됩니다. Tyr 1021에서의 인산화는 Grb2 및 PLC-γ와 같은 신호 분자를 모집하여 세포 증식, 이동, 생존 및 혈관신생을 조절하는 경로를 시작합니다. 이 인산화는 조직 복구 및 발달에서 PDGFRβ의 역할에 필수적이며, 그 조절 장애는 암 및 섬유증과 같은 병리학적 상태에 기여하므로 잠재적인 치료 표적이 됩니다.

사용 정보

응용

WB

희석

WB

1:1000

반응성

Human, Mouse, Rat

출처

Rabbit Monoclonal Antibody

MW

190 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 1351. 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 60s 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

1351. 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 60s is recommended)

참조

https://pubmed.ncbi.nlm.nih.gov/20534510/
https://pubmed.ncbi.nlm.nih.gov/23906712/

적용 데이터

WB

Selleck 검증

Lane 1: NIH/3T3
Lane 2: NIH/3T3 (PDGF-BB-treated)