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생물학적 설명

특이성	GPD2 Antibody [G9G15]는 총 GPD2 단백질의 내인성 수준을 인식합니다.
배경	GPD2 (미토콘드리아 글리세롤-3-인산 Dehydrogenase)는 미토콘드리아 내막의 바깥 표면에 위치한 플라빈 아데닌 디뉴클레오티드(FAD) 의존성 효소로, 글리세롤 인산 셔틀(GPS)의 핵심 구성 요소로 기능합니다. 세포질 GPD1과 함께 GPD2는 글리세롤-3-인산(G3P)을 디하이드록시아세톤 인산(DHAP)으로 전환하고 FAD를 FADH2로 환원시켜 NADH로부터 미토콘드리아 전자 전달 사슬로 전자를 전달하는 것을 촉진하며, FADH2는 보효소 Q에 전자를 제공합니다. 염색체 2q24.1에 위치한 GPD2 유전자는 갈색 지방 조직, 뇌, 고환과 같이 Metabolism적으로 활발한 조직에서 높게 발현되는 보존된 727-아미노산 단백질을 암호화하며, 세 가지 프로모터(A, B, C)에 의해 조절됩니다. GPD2는 포도당 산화, ATP 생산 및 히스톤 아세틸화에 중요하며, 이를 통해 염증성 유전자 발현, 열 발생 및 갑상선 호르몬 매개 에너지 Metabolism에 영향을 미칩니다. 또한 증식하는 세포의 Metabolism 요구를 지원함으로써 주로 종양 촉진 인자로 작용합니다.

특이성

GPD2 Antibody [G9G15]는 총 GPD2 단백질의 내인성 수준을 인식합니다.

배경

GPD2 (미토콘드리아 글리세롤-3-인산 Dehydrogenase)는 미토콘드리아 내막의 바깥 표면에 위치한 플라빈 아데닌 디뉴클레오티드(FAD) 의존성 효소로, 글리세롤 인산 셔틀(GPS)의 핵심 구성 요소로 기능합니다. 세포질 GPD1과 함께 GPD2는 글리세롤-3-인산(G3P)을 디하이드록시아세톤 인산(DHAP)으로 전환하고 FAD를 FADH2로 환원시켜 NADH로부터 미토콘드리아 전자 전달 사슬로 전자를 전달하는 것을 촉진하며, FADH2는 보효소 Q에 전자를 제공합니다. 염색체 2q24.1에 위치한 GPD2 유전자는 갈색 지방 조직, 뇌, 고환과 같이 Metabolism적으로 활발한 조직에서 높게 발현되는 보존된 727-아미노산 단백질을 암호화하며, 세 가지 프로모터(A, B, C)에 의해 조절됩니다. GPD2는 포도당 산화, ATP 생산 및 히스톤 아세틸화에 중요하며, 이를 통해 염증성 유전자 발현, 열 발생 및 갑상선 호르몬 매개 에너지 Metabolism에 영향을 미칩니다. 또한 증식하는 세포의 Metabolism 요구를 지원함으로써 주로 종양 촉진 인자로 작용합니다.

사용 정보

응용

WB, IP, FCM

희석

WB	IP	FCM
1:1000-1:10000	1:30-1:50	1:310

반응성

Human

출처

Rabbit Monoclonal Antibody

MW

81 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: 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 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: 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/31384058/
https://pubmed.ncbi.nlm.nih.gov/38689091/

적용 데이터

WB

Selleck 검증

Lane 1: HeLa, Lane 2: MCF-7, Lane 3: NIH:OVCAR-3, Lane 4: U87-MG