데이터시트

생물학적 설명

특이성	Phospho-DRP1 (Ser637) Antibody [J22A19]는 Ser637에서 인산화된 경우에만 내인성 DRP1 단백질 수준을 인식합니다. 쥐의 경우, DRP1의 인산화 부위는 Ser 656입니다.
배경	Dynamin-related protein 1 (Drp1)은 미토콘드리아 분열을 주로 매개하는 다이내민(Dynamin) 슈퍼패밀리의 대형 GTPase이며, 미토파지 및 미토콘드리아 역학을 조절하여 세포 건강을 보장합니다. 구조적으로 Drp1은 N-말단 GTPase 도메인, 나선형 중간 도메인, GTPase 도메인으로의 분자내 역접힘을 가진 GED 도메인, 그리고 막 결합을 위한 힌지 역할을 하는 가변 도메인 등 네 가지 주요 도메인으로 구성됩니다. 활성 Drp1은 미토콘드리아 외막으로 이동하여 분열을 유도하는데, 이는 미토콘드리아 기능 유지에 필수적인 과정입니다. 특히 PKA에 의한 Ser-637에서의 Drp1 인산화는 GTP-GED 상호작용을 방해하여 GTPase 활성을 감소시키고, 이는 분열을 억제하며 세포 주기A의B1 다른 단계에서 Drp1 활성을 조절합니다. 인산화는 Drp1 활성 조절에 중요합니다. Ser637에서의 인산화는 Drp1이 세포질에서 미토콘드리아 외막으로 이동하는 것을 억제합니다. 미토콘드리아 분열 동안 Drp1 인산화에 관여하는 주요 키나아제는 PGAM5, AMPK, MAPK, 그리고 Cdk1/cyclin B1을 포함합니다.

특이성

Phospho-DRP1 (Ser637) Antibody [J22A19]는 Ser637에서 인산화된 경우에만 내인성 DRP1 단백질 수준을 인식합니다. 쥐의 경우, DRP1의 인산화 부위는 Ser 656입니다.

배경

Dynamin-related protein 1 (Drp1)은 미토콘드리아 분열을 주로 매개하는 다이내민(Dynamin) 슈퍼패밀리의 대형 GTPase이며, 미토파지 및 미토콘드리아 역학을 조절하여 세포 건강을 보장합니다. 구조적으로 Drp1은 N-말단 GTPase 도메인, 나선형 중간 도메인, GTPase 도메인으로의 분자내 역접힘을 가진 GED 도메인, 그리고 막 결합을 위한 힌지 역할을 하는 가변 도메인 등 네 가지 주요 도메인으로 구성됩니다. 활성 Drp1은 미토콘드리아 외막으로 이동하여 분열을 유도하는데, 이는 미토콘드리아 기능 유지에 필수적인 과정입니다. 특히 PKA에 의한 Ser-637에서의 Drp1 인산화는 GTP-GED 상호작용을 방해하여 GTPase 활성을 감소시키고, 이는 분열을 억제하며 세포 주기A의B1 다른 단계에서 Drp1 활성을 조절합니다. 인산화는 Drp1 활성 조절에 중요합니다. Ser637에서의 인산화는 Drp1이 세포질에서 미토콘드리아 외막으로 이동하는 것을 억제합니다. 미토콘드리아 분열 동안 Drp1 인산화에 관여하는 주요 키나아제는 PGAM5, AMPK, MAPK, 그리고 Cdk1/cyclin B1을 포함합니다.

사용 정보

응용

WB

희석

WB

1:1000

반응성

Rat

출처

Rabbit Monoclonal Antibody

MW

78-82 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, Phosphatase 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, Phosphatase 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, Phosphatase 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 ( recommending 5% BSA 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 1205. 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, Phosphatase 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, Phosphatase 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, Phosphatase 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 ( recommending 5% BSA 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

1205. 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/32913266/
https://pubmed.ncbi.nlm.nih.gov/32433544/

적용 데이터

WB

Selleck 검증

Lane 1: PC-12
Lane 2: PC-12 (Forskolin, 20 μM, 1 h)