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생물학적 설명

특이성	Phospho-MEK1 (Ser298) Antibody [L22P14]는 Ser298에서 인산화될 때에만 총 MEK1 단백질의 내인성 수준을 감지합니다.
배경	Phospho-MEK1 (Ser298)은 MAPK/ERK 캐스케이드에 있는 이중 특이성 키나아제인 MEK1을 의미하며, PAK1에 의해 세린 298에서 인산화되어 Raf 매개 활성화 루프 세린 218/222의 인산화를 강화하여 세포 성장, 분화 및 접착 반응을 위해 ERK1/2로 신호 전달을 유도합니다. MEK1은 N- 및 C-엽, 프롤린이 풍부한 모티프, 그리고 B-Raf의 Arg662와 같은 상류 조절자와 상호 작용하여 활성화 동안 A-루프 재배치 및 인산화 유연성을 촉진하는 A-루프 근처의 Asp217과 같은 주요 잔기를 포함하는 전형적인 키나아제 도메인을 특징으로 합니다. PAK1에 의한 Ser298 인산화는 인테그린 및 성장 인자 신호 전달을 위한 중요한 수렴점 역할을 하며, FAK/Src 의존성을 통해 Phospho-MEK1을 주변 접착 부위에 국소화하고, MEK1-ERK 복합체 형성을 촉진하고, MAPK 활성화 및 세포 확산과 같은 피브로넥틴 자극 반응을 촉진합니다. 반면, 이 부위가 없는 돌연변이(S298A)는 NIH/3T3 또는 다른 세포에서 ERK 인산화 및 경로 효율성을 손상시킵니다. 이 변형은 혈청 비의존성 기저 인산화와는 다른 접착 조절 방식으로, MEK2에 대한 교차 반응성 없이 변형 또는 분화를 지원합니다.

특이성

Phospho-MEK1 (Ser298) Antibody [L22P14]는 Ser298에서 인산화될 때에만 총 MEK1 단백질의 내인성 수준을 감지합니다.

배경

Phospho-MEK1 (Ser298)은 MAPK/ERK 캐스케이드에 있는 이중 특이성 키나아제인 MEK1을 의미하며, PAK1에 의해 세린 298에서 인산화되어 Raf 매개 활성화 루프 세린 218/222의 인산화를 강화하여 세포 성장, 분화 및 접착 반응을 위해 ERK1/2로 신호 전달을 유도합니다. MEK1은 N- 및 C-엽, 프롤린이 풍부한 모티프, 그리고 B-Raf의 Arg662와 같은 상류 조절자와 상호 작용하여 활성화 동안 A-루프 재배치 및 인산화 유연성을 촉진하는 A-루프 근처의 Asp217과 같은 주요 잔기를 포함하는 전형적인 키나아제 도메인을 특징으로 합니다. PAK1에 의한 Ser298 인산화는 인테그린 및 성장 인자 신호 전달을 위한 중요한 수렴점 역할을 하며, FAK/Src 의존성을 통해 Phospho-MEK1을 주변 접착 부위에 국소화하고, MEK1-ERK 복합체 형성을 촉진하고, MAPK 활성화 및 세포 확산과 같은 피브로넥틴 자극 반응을 촉진합니다. 반면, 이 부위가 없는 돌연변이(S298A)는 NIH/3T3 또는 다른 세포에서 ERK 인산화 및 경로 효율성을 손상시킵니다. 이 변형은 혈청 비의존성 기저 인산화와는 다른 접착 조절 방식으로, MEK2에 대한 교차 반응성 없이 변형 또는 분화를 지원합니다.

사용 정보

응용

WB, IP

희석

WB	IP
1:1000	1:50

반응성

Human, Mouse, Rat

출처

Rabbit Monoclonal Antibody

MW

45 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. 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, Phosphatase 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, Phosphatase 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 ( 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 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, Phosphatase 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, Phosphatase 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, Phosphatase 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 ( 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

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/17314031/
https://pubmed.ncbi.nlm.nih.gov/12876277/

적용 데이터

WB

Selleck 검증

Lane 1: MDA-MB-231, Lane 2: MDA-MB-231 (phosphatase treated), Lane 3: NIH/3T3, Lane 4: NIH/3T3 (phosphatase treated)