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특이성	PINK1 Antibody [B2H2]는 총 PINK1 단백질의 내인성 수준을 인식합니다.
배경	"PTEN-induced putative kinase 1 (PINK1)은 미토콘드리아 세린/트레오닌 키나아제로서 미토콘드리아 기능 및 무결성 유지뿐만 아니라 미토콘드리아로부터의 사이토크롬 c 방출 최소화에 중요한 역할을 합니다. PINK1은 조기 발병 파킨슨병(PD) 발병과 밀접하게 관련되어 있습니다. 이는 파킨(Parkin) 상류에서 작용하여 미토파지(mitophagy) 과정을 통해 손상된 미토콘드리아 제거를 촉진합니다. 신경퇴행 및 미토파지 관여 외에도 PINK1은 세포 대사 조절, 암 진행 및 염증을 포함한 여러 다른 역할을 합니다. PINK1은 JNK 및 ERK 신호 경로 활성화를 통해 간 인슐린 저항성(IR)에 기여하는 것으로 나타났습니다. 또한 미토콘드리아 유래 소포(MDVs) 형성을 억제하고 염증 반응 동안 미토콘드리아 항원 제시(MitAP)를 제한합니다. 또한 PINK1은 특히 B형 간염(HBV) 또는 C형 간염(HCV) 감염과 관련하여 간 염증과 연관되어 있습니다. PINK1의 키나아제 도메인은 RLR 매개 1형 인터페론 및 전염증성 사이토카인 생산을 강화하는 데 필수적입니다. "

특이성

PINK1 Antibody [B2H2]는 총 PINK1 단백질의 내인성 수준을 인식합니다.

배경

"PTEN-induced putative kinase 1 (PINK1)은 미토콘드리아 세린/트레오닌 키나아제로서 미토콘드리아 기능 및 무결성 유지뿐만 아니라 미토콘드리아로부터의 사이토크롬 c 방출 최소화에 중요한 역할을 합니다. PINK1은 조기 발병 파킨슨병(PD) 발병과 밀접하게 관련되어 있습니다. 이는 파킨(Parkin) 상류에서 작용하여 미토파지(mitophagy) 과정을 통해 손상된 미토콘드리아 제거를 촉진합니다. 신경퇴행 및 미토파지 관여 외에도 PINK1은 세포 대사 조절, 암 진행 및 염증을 포함한 여러 다른 역할을 합니다. PINK1은 JNK 및 ERK 신호 경로 활성화를 통해 간 인슐린 저항성(IR)에 기여하는 것으로 나타났습니다. 또한 미토콘드리아 유래 소포(MDVs) 형성을 억제하고 염증 반응 동안 미토콘드리아 항원 제시(MitAP)를 제한합니다. 또한 PINK1은 특히 B형 간염(HBV) 또는 C형 간염(HCV) 감염과 관련하여 간 염증과 연관되어 있습니다. PINK1의 키나아제 도메인은 RLR 매개 1형 인터페론 및 전염증성 사이토카인 생산을 강화하는 데 필수적입니다. "

사용 정보

응용

WB, IP

희석

WB	IP
1:1000	1:50

반응성

Human

출처

Rabbit Monoclonal Antibody

MW

60, 50 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 1047. 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: 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

1047. 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/31139191/

적용 데이터

WB

Selleck 검증

Lane 1: HeLa
Lane 2: HeLa (CCCP, 10 μM, 24 h)