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생물학적 설명

특이성	PDK4 Antibody [C15G7]는 총 PDK4 단백질의 내인성 수준을 검출합니다.
배경	피루브산 탈수소효소 키나아제 4(PDK4)는 PDK 계열에 속하는 미토콘드리아 기질 효소로, 포도당 및 지방산 대사 조절에 필수적입니다. 구조적으로 PDK4는 인산화 활성을 담당하는 히스티딘 키나아제 도메인, 국소화를 위한 미토콘드리아 표적 서열 및 단백질-단백질 상호작용을 위한 조절 영역을 포함합니다. 심장, 골격근 및 간에서 높게 발현되며, 글루코코르티코이드, 레티노산, 프로락틴(STAT5를 통해), 장쇄 지방산 및 피브레이트와 같은 PPARα 작용제에 의해 발현이 상향 조절되는 반면, 인슐린에 의해 억제됩니다. PDK4는 피루브산 탈수소효소 복합체(PDC)를 인산화하고 억제하여 피루브산의 아세틸-CoA 전환을 감소시키고, 이로써 금식 또는 에너지 결핍 시 탄수화물 산화에서 지방산 활용으로 대사를 전환합니다. 조절 이상 PDK4 발현은 제2형 당뇨병, 인슐린 저항성 및 심혈관 질환을 포함한 대사 장애와 관련이 있으며, 자가포식 억제 및 대사 재프로그래밍을 통해 혈관 석회화 및 죽상동맥경화증에 기여합니다. PDK4의 적응 조절은 에너지 항상성 및 대사 유연성에 필수적입니다.

특이성

PDK4 Antibody [C15G7]는 총 PDK4 단백질의 내인성 수준을 검출합니다.

배경

피루브산 탈수소효소 키나아제 4(PDK4)는 PDK 계열에 속하는 미토콘드리아 기질 효소로, 포도당 및 지방산 대사 조절에 필수적입니다. 구조적으로 PDK4는 인산화 활성을 담당하는 히스티딘 키나아제 도메인, 국소화를 위한 미토콘드리아 표적 서열 및 단백질-단백질 상호작용을 위한 조절 영역을 포함합니다. 심장, 골격근 및 간에서 높게 발현되며, 글루코코르티코이드, 레티노산, 프로락틴(STAT5를 통해), 장쇄 지방산 및 피브레이트와 같은 PPARα 작용제에 의해 발현이 상향 조절되는 반면, 인슐린에 의해 억제됩니다. PDK4는 피루브산 탈수소효소 복합체(PDC)를 인산화하고 억제하여 피루브산의 아세틸-CoA 전환을 감소시키고, 이로써 금식 또는 에너지 결핍 시 탄수화물 산화에서 지방산 활용으로 대사를 전환합니다. 조절 이상 PDK4 발현은 제2형 당뇨병, 인슐린 저항성 및 심혈관 질환을 포함한 대사 장애와 관련이 있으며, 자가포식 억제 및 대사 재프로그래밍을 통해 혈관 석회화 및 죽상동맥경화증에 기여합니다. PDK4의 적응 조절은 에너지 항상성 및 대사 유연성에 필수적입니다.

사용 정보

응용

WB, IP, FCM

희석

WB	IP	FCM
1:1000	1:30	1:60

반응성

Mouse, Rat

출처

Rabbit Monoclonal Antibody

MW

47 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 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 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 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 1375. 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 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 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 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

1375. 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/19703515/
https://pubmed.ncbi.nlm.nih.gov/33203874/

적용 데이터

WB

Selleck 검증

Lane 1: Mouse heart
Lane 2: Rat heart
Lane 3: C2C12
Lane 4: C6