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생물학적 설명

특이성	POLG Antibody [M21J18]는 총 POLG 단백질의 내인성 수준을 검출합니다.
배경	미토콘드리아 DNA(mtDNA)는 미토콘드리아 내부 기질 내의 개별 핵양체(nucleoid)에 존재하며, 각 핵양체는 일반적으로 한두 개의 mtDNA 사본을 포함합니다. mtDNA 복제는 DNA 중합효소 γ(pol γ), 미토콘드리아 단일 가닥 DNA 결합 단백질, Twinkle mtDNA 헬리카제, 그리고 토포이소머라제 및 RNase H를 포함하는 핵심 복제 단백질로 구성된 특수 복제체(replisome)에 의해 수행됩니다. 인간 pol γ는 두 개의 서브유닛으로 구성된 이종이합체입니다. 15q25 염색체 좌위의 POLG 유전자에 의해 암호화되는 140 kDa 촉매 서브유닛인 POLG는 DNA 중합효소, 3′→5′ 엑소뉴클레아제, 5′-데옥시리보스 인산(5′-dRP) 리아제 활성을 가집니다. 이는 아미노-말단 엑소뉴클레아제 도메인이 링커에 의해 카르복시-말단 중합효소 도메인에 연결되어 있습니다. 17q24 염색체 좌위의 POLG2에 의해 암호화되는 55 kDa 보조 서브유닛인 POLG2는 이합체를 형성하고 촉매 서브유닛의 DNA에 대한 친화도를 증가시켜 중합효소 진행성을 향상시킵니다. POLG의 돌연변이는 유전성 미토콘드리아 질환의 주요 원인이며, 인구의 최대 2%가 병원성 변이체를 가지고 있습니다. 이러한 돌연변이는 유아기 mtDNA 결핍 증후군 또는 mtDNA 결실로 인한 후기 발병 질환으로 이어질 수 있습니다. POLG 관련 질환은 유아기부터 성인 후기까지 발병하는 중첩되는 표현형의 연속체를 형성합니다. POLG 돌연변이와 관련된 여섯 가지 주요 임상 증후군은 알퍼스–후텐로허 증후군(가장 심각한 표현형 중 하나), 소아기 근뇌간간병증 스펙트럼, 근간대성 간질 근병증 감각 실조증, 실조증 신경병증 스펙트럼, 상염색체 열성 진행성 외안근 마비, 상염색체 우성 진행성 외안근 마비입니다.

특이성

POLG Antibody [M21J18]는 총 POLG 단백질의 내인성 수준을 검출합니다.

배경

미토콘드리아 DNA(mtDNA)는 미토콘드리아 내부 기질 내의 개별 핵양체(nucleoid)에 존재하며, 각 핵양체는 일반적으로 한두 개의 mtDNA 사본을 포함합니다. mtDNA 복제는 DNA 중합효소 γ(pol γ), 미토콘드리아 단일 가닥 DNA 결합 단백질, Twinkle mtDNA 헬리카제, 그리고 토포이소머라제 및 RNase H를 포함하는 핵심 복제 단백질로 구성된 특수 복제체(replisome)에 의해 수행됩니다. 인간 pol γ는 두 개의 서브유닛으로 구성된 이종이합체입니다. 15q25 염색체 좌위의 POLG 유전자에 의해 암호화되는 140 kDa 촉매 서브유닛인 POLG는 DNA 중합효소, 3′→5′ 엑소뉴클레아제, 5′-데옥시리보스 인산(5′-dRP) 리아제 활성을 가집니다. 이는 아미노-말단 엑소뉴클레아제 도메인이 링커에 의해 카르복시-말단 중합효소 도메인에 연결되어 있습니다. 17q24 염색체 좌위의 POLG2에 의해 암호화되는 55 kDa 보조 서브유닛인 POLG2는 이합체를 형성하고 촉매 서브유닛의 DNA에 대한 친화도를 증가시켜 중합효소 진행성을 향상시킵니다. POLG의 돌연변이는 유전성 미토콘드리아 질환의 주요 원인이며, 인구의 최대 2%가 병원성 변이체를 가지고 있습니다. 이러한 돌연변이는 유아기 mtDNA 결핍 증후군 또는 mtDNA 결실로 인한 후기 발병 질환으로 이어질 수 있습니다. POLG 관련 질환은 유아기부터 성인 후기까지 발병하는 중첩되는 표현형의 연속체를 형성합니다. POLG 돌연변이와 관련된 여섯 가지 주요 임상 증후군은 알퍼스–후텐로허 증후군(가장 심각한 표현형 중 하나), 소아기 근뇌간간병증 스펙트럼, 근간대성 간질 근병증 감각 실조증, 실조증 신경병증 스펙트럼, 상염색체 열성 진행성 외안근 마비, 상염색체 우성 진행성 외안근 마비입니다.

사용 정보

응용

WB

희석

WB

1:1000 - 1:10000

반응성

Mouse, Rat, Human

출처

Rabbit Monoclonal Antibody

MW

140 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 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 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 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: 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 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. (Exposure time of at least 120s is recommended)

실험 방법

WB

Experimental Protocol:

Sample preparation

1. Tissue: Lyse the tissue sample by adding an appropriate volume of ice-cold RIPA 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 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 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: 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

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. (Exposure time of at least 120s is recommended)

참조

https://pubmed.ncbi.nlm.nih.gov/30451971/

적용 데이터

WB

Selleck 검증

Lane 1: HEK-293T, Lane 2: HepG2