데이터시트

생물학적 설명

특이성	Mitofusin 1 Antibody [D5A17]는 총 Mitofusin-1 단백질의 내인성 수준을 검출합니다.
배경	미토푸신 1(MFN1)은 외측 미토콘드리아 막에 위치한 다이내민 관련 GTPase 계열의 핵심 구성원으로, 미토콘드리아 네트워크 무결성, 에너지 항상성 및 세포 생존을 유지하는 데 필수적인 미토콘드리아 융합에 필수적입니다. MFN1은 GTP 결합 및 가수분해를 가능하게 하는 N-말단 GTPase 도메인, 인접한 미토콘드리아 간의 이합체화 및 연결을 매개하는 코일형 코일 구조를 형성하는 두 개의 헵타드 반복 영역(HR1 및 HR2), 그리고 단백질을 외막 내에 고정시키는 두 개의 막관통 나선형 구조를 포함합니다. 확장된 HR2는 인접 미토콘드리아의 MFN1 또는 MFN2와의 직접적인 상호작용을 촉진하여 효율적인 융합을 유도합니다. MFN1은 MFN2 및 OPA1과 긴밀하게 협력하여 미토콘드리아 형태를 보존하고, 미토콘드리아 DNA(mtDNA)를 안정화하며, ATP 생성을 조절하고, 세포자멸사 경로를 지원하며, 스트레스 하에서 대사 적응을 가능하게 합니다. 또한 기능 장애 미토콘드리아의 축적을 방지하는 미토파지를 촉진하며, 배아 발생 및 신경 발달에 중요합니다. MFN1의 손실 또는 기능 장애는 미토콘드리아 역학을 손상시켜 에너지 결핍, 신경 퇴행 및 대사 질환을 유발합니다. MFN1 돌연변이는 MFN2의 돌연변이보다 덜 흔하지만, 결핍은 샤르코-마리-투스 유형 2A와 유사한 신경병증, 근 위축 및 신경염증 상태를 악화시킵니다. 변형된 MFN1 발현은 파킨슨병, 알츠하이머병 및 일부 암과 같은 질병에서 관찰됩니다.

특이성

Mitofusin 1 Antibody [D5A17]는 총 Mitofusin-1 단백질의 내인성 수준을 검출합니다.

배경

미토푸신 1(MFN1)은 외측 미토콘드리아 막에 위치한 다이내민 관련 GTPase 계열의 핵심 구성원으로, 미토콘드리아 네트워크 무결성, 에너지 항상성 및 세포 생존을 유지하는 데 필수적인 미토콘드리아 융합에 필수적입니다. MFN1은 GTP 결합 및 가수분해를 가능하게 하는 N-말단 GTPase 도메인, 인접한 미토콘드리아 간의 이합체화 및 연결을 매개하는 코일형 코일 구조를 형성하는 두 개의 헵타드 반복 영역(HR1 및 HR2), 그리고 단백질을 외막 내에 고정시키는 두 개의 막관통 나선형 구조를 포함합니다. 확장된 HR2는 인접 미토콘드리아의 MFN1 또는 MFN2와의 직접적인 상호작용을 촉진하여 효율적인 융합을 유도합니다. MFN1은 MFN2 및 OPA1과 긴밀하게 협력하여 미토콘드리아 형태를 보존하고, 미토콘드리아 DNA(mtDNA)를 안정화하며, ATP 생성을 조절하고, 세포자멸사 경로를 지원하며, 스트레스 하에서 대사 적응을 가능하게 합니다. 또한 기능 장애 미토콘드리아의 축적을 방지하는 미토파지를 촉진하며, 배아 발생 및 신경 발달에 중요합니다. MFN1의 손실 또는 기능 장애는 미토콘드리아 역학을 손상시켜 에너지 결핍, 신경 퇴행 및 대사 질환을 유발합니다. MFN1 돌연변이는 MFN2의 돌연변이보다 덜 흔하지만, 결핍은 샤르코-마리-투스 유형 2A와 유사한 신경병증, 근 위축 및 신경염증 상태를 악화시킵니다. 변형된 MFN1 발현은 파킨슨병, 알츠하이머병 및 일부 암과 같은 질병에서 관찰됩니다.

사용 정보

응용

WB, IP

희석

WB	IP
1:1000	1:30

반응성

Mouse, Rat

출처

Rabbit Monoclonal Antibody

MW

84 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. 2. Adherent cell: Aspirate the culture medium and wash the cells with ice-cold PBS twice. Lyse the cells by adding an appropriate volume of RIPA/NP-40 Lysis Buffer (containing Protease Inhibitor Cocktail) and put the sample on ice for 5 min. 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. Lyse the cells by adding an appropriate volume of RIPA/NP-40 Lysis Buffer (containing Protease Inhibitor Cocktail) and put the sample on ice for 5 min. 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 1. 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.

2. Adherent cell: Aspirate the culture medium and wash the cells with ice-cold PBS twice. Lyse the cells by adding an appropriate volume of RIPA/NP-40 Lysis Buffer (containing Protease Inhibitor Cocktail) and put the sample on ice for 5 min.

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. Lyse the cells by adding an appropriate volume of RIPA/NP-40 Lysis Buffer (containing Protease Inhibitor Cocktail) and put the sample on ice for 5 min.

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

1. 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/30647902/
https://pubmed.ncbi.nlm.nih.gov/27920125/

적용 데이터

WB

Selleck 검증

Lane 1: NIH/3T3, Lane 2: Neuro-2a, Lane 3: Mouse brain, Lane 4: Rat brain