데이터시트

생물학적 설명

특이성	Angiotensin II Type 2 Receptor Antibody [F9C2]는 총 안지오텐신 II 유형 2 수용체 단백질의 내인성 수준을 인식합니다.
배경	GPCR & G Protein 계열의 구성원인 Angiotensin II Type 2 Receptor (AT2R)는 자극성(Gαs) 또는 억제성(Gαi/o) 단백질 및 AT2R 결합 단백질을 포함하는 G 단백질 비의존적 경로와 연결되어 있습니다. AT2R의 활성화는 신호 전달 경로에서 중요한 역할을 하는 SHP-1(SH2 도메인 함유 포스파타제 1), MKP-1(미토겐 활성화 단백질 키나제 포스파타제 1) 및 PP2A(단백질 포스파타제 2A)와 같은 효소를 활성화합니다. AT2R은 염증 감소, 심실 기능 개선, 흉터 조직 감소, 세포자멸사 조절 및 세포 생존 경로를 통한 유해한 심장 리모델링 방지를 통해 심혈관 및 신장 보호에 필수적입니다. AT2R의 부재는 심부전 악화로 이어지며, 적당한 과발현은 손상 후 심장 기능을 향상시킵니다. AT2R의 활성화는 또한 섬유증 감소, 사구체 여과율(GFR) 개선, 염증 감소와 관련이 있으며, 특히 당뇨병성 신병증과 같은 신장 질환에 유익합니다. TNF-α 및 IL-6와 같은 사이토카인을 낮추고 IL-10을 증가시켜 항염증 효과를 나타내며, 이는 심장 및 신장 건강을 위한 면역 세포 조절에 도움이 됩니다. MasR과의 상호작용은 바이러스로 인한 ACE2 하향 조절에 잠재적으로 대응하여 유리한 Ang (1–7)/MasR 신호 전달 경로를 복원함으로써 SARS-CoV-2 치료에 대한 가능성을 제시합니다.

특이성

Angiotensin II Type 2 Receptor Antibody [F9C2]는 총 안지오텐신 II 유형 2 수용체 단백질의 내인성 수준을 인식합니다.

배경

GPCR & G Protein 계열의 구성원인 Angiotensin II Type 2 Receptor (AT2R)는 자극성(Gαs) 또는 억제성(Gαi/o) 단백질 및 AT2R 결합 단백질을 포함하는 G 단백질 비의존적 경로와 연결되어 있습니다. AT2R의 활성화는 신호 전달 경로에서 중요한 역할을 하는 SHP-1(SH2 도메인 함유 포스파타제 1), MKP-1(미토겐 활성화 단백질 키나제 포스파타제 1) 및 PP2A(단백질 포스파타제 2A)와 같은 효소를 활성화합니다. AT2R은 염증 감소, 심실 기능 개선, 흉터 조직 감소, 세포자멸사 조절 및 세포 생존 경로를 통한 유해한 심장 리모델링 방지를 통해 심혈관 및 신장 보호에 필수적입니다. AT2R의 부재는 심부전 악화로 이어지며, 적당한 과발현은 손상 후 심장 기능을 향상시킵니다. AT2R의 활성화는 또한 섬유증 감소, 사구체 여과율(GFR) 개선, 염증 감소와 관련이 있으며, 특히 당뇨병성 신병증과 같은 신장 질환에 유익합니다. TNF-α 및 IL-6와 같은 사이토카인을 낮추고 IL-10을 증가시켜 항염증 효과를 나타내며, 이는 심장 및 신장 건강을 위한 면역 세포 조절에 도움이 됩니다. MasR과의 상호작용은 바이러스로 인한 ACE2 하향 조절에 잠재적으로 대응하여 유리한 Ang (1–7)/MasR 신호 전달 경로를 복원함으로써 SARS-CoV-2 치료에 대한 가능성을 제시합니다.

사용 정보

응용

WB, IP

희석

WB	IP
1:1000-1:10000	1:20

반응성

Human, Mouse, Rat

출처

Rabbit Monoclonal Antibody

MW

41 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: 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 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 1059. 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.

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: 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 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

1059. 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.

참조

https://pubmed.ncbi.nlm.nih.gov/33840201/

적용 데이터

WB

Selleck 검증

Lane 1: HepG2
Lane 2: Human heart
Lane 3: Mouse heart
Lane 4: Rat heart