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생물학적 설명

특이성	VE-cadherin Antibody [C2J1]는 총 VE-cadherin 단백질의 내인성 수준을 인식합니다.
배경	VE-cadherin은 혈관 내피 카드헤린 또는 CD144로도 알려져 있으며, 주로 내피 세포에 발현되는 중요한 막관통 단백질로, 혈관 투과성과 완전성을 조절하는 세포-세포 접합을 유지하는 데 중요한 역할을 합니다. 카드헤린 슈퍼패밀리의 구성원으로서, VE-cadherin은 인접한 VE-cadherin 분자와의 동종결합을 담당하는 세포외 도메인, 단일 막관통 도메인, 그리고 β-카테닌 및 p120-카테닌과 같은 카테닌과 상호작용하는 세포내 도메인으로 구성된 독특한 구조를 특징으로 합니다. 이 상호작용은 VE-cadherin을 액틴 세포골격에 고정시켜 내피 세포 간의 강력한 접착을 촉진하고 내피 장벽의 안정성에 필수적인 접착 접합을 형성합니다. VE-cadherin은 혈관 구조의 성숙 및 안정화를 조절함으로써 혈관신생에 중요합니다. 또한 내피를 가로지르는 백혈구 이동을 조절하여 염증에 중요한 역할을 합니다. 염증 반응 동안 VE-cadherin의 인산화는 세포-세포 접착을 약화시켜 면역 세포가 손상 또는 감염 부위로 이동할 수 있도록 합니다. VE-cadherin 활성의 조절은 Src와 같은 키나아제에 의한 인산화를 포함한 여러 메커니즘을 포함하며, 이는 VEGF와 같은 성장 인자에 대한 반응으로 접착 특성 및 투과성에 영향을 미칩니다. VE-cadherin의 조절 이상은 암 및 염증성 질환을 유발합니다.

특이성

VE-cadherin Antibody [C2J1]는 총 VE-cadherin 단백질의 내인성 수준을 인식합니다.

배경

VE-cadherin은 혈관 내피 카드헤린 또는 CD144로도 알려져 있으며, 주로 내피 세포에 발현되는 중요한 막관통 단백질로, 혈관 투과성과 완전성을 조절하는 세포-세포 접합을 유지하는 데 중요한 역할을 합니다. 카드헤린 슈퍼패밀리의 구성원으로서, VE-cadherin은 인접한 VE-cadherin 분자와의 동종결합을 담당하는 세포외 도메인, 단일 막관통 도메인, 그리고 β-카테닌 및 p120-카테닌과 같은 카테닌과 상호작용하는 세포내 도메인으로 구성된 독특한 구조를 특징으로 합니다. 이 상호작용은 VE-cadherin을 액틴 세포골격에 고정시켜 내피 세포 간의 강력한 접착을 촉진하고 내피 장벽의 안정성에 필수적인 접착 접합을 형성합니다. VE-cadherin은 혈관 구조의 성숙 및 안정화를 조절함으로써 혈관신생에 중요합니다. 또한 내피를 가로지르는 백혈구 이동을 조절하여 염증에 중요한 역할을 합니다. 염증 반응 동안 VE-cadherin의 인산화는 세포-세포 접착을 약화시켜 면역 세포가 손상 또는 감염 부위로 이동할 수 있도록 합니다. VE-cadherin 활성의 조절은 Src와 같은 키나아제에 의한 인산화를 포함한 여러 메커니즘을 포함하며, 이는 VEGF와 같은 성장 인자에 대한 반응으로 접착 특성 및 투과성에 영향을 미칩니다. VE-cadherin의 조절 이상은 암 및 염증성 질환을 유발합니다.

사용 정보

응용

WB, IP, FCM, ELISA

희석

WB	IP	FCM
1:100-1:1000	1:50	1:50

반응성

Human, Mouse, Rat, Porcine

출처

Mouse Monoclonal Antibody

MW

130 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)，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), 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), 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 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 1387. 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)，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), 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), 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 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

1387. 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/18162609/
https://pubmed.ncbi.nlm.nih.gov/20708398/

적용 데이터

WB

Selleck 검증

Lane 1: HUV-EC-C
Lane 2: Human placenta