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Descripción biológica

Especificidad	Phospho-MEK1 (Ser298) Antibody [L22P14] detecta niveles endógenos de la proteína MEK1 total solo cuando está fosforilada en Ser298
Antecedentes	Phospho-MEK1 (Ser298) se refiere a MEK1, una quinasa de doble especificidad en la cascada MAPK/ERK, fosforilada en la serina 298 por PAK1, lo que mejora la fosforilación mediada por Raf de sus serinas del bucle de activación 218/222 para impulsar la transducción de señales a ERK1/2 para el crecimiento celular, la diferenciación y las respuestas de adhesión. MEK1 presenta un dominio quinasa típico con lóbulos N y C, motivos ricos en prolina y residuos clave, como Asp217 cerca del bucle A, que interactúan con reguladores río arriba, como Arg662 de B-Raf, para facilitar el reposicionamiento del bucle A y la flexibilidad de fosforilación durante la activación. La fosforilación de Ser298 por PAK1 sirve como una convergencia crítica para la señalización de integrinas y factores de crecimiento, localizando phospho-MEK1 en sitios de adhesión periféricos a través de la dependencia de FAK/Src, promoviendo la formación del complejo MEK1-ERK, la activación de MAPK y las respuestas estimuladas por fibronectina como la propagación celular, mientras que los mutantes que carecen de este sitio (S298A) alteran la fosforilación de ERK y la eficiencia de la vía en células NIH/3T3 u otras células. Esta modificación está regulada por la adhesión, es distinta de la fosforilación basal independiente del suero y apoya la transformación o diferenciación sin reactividad cruzada con MEK2.

Especificidad

Phospho-MEK1 (Ser298) Antibody [L22P14] detecta niveles endógenos de la proteína MEK1 total solo cuando está fosforilada en Ser298

Antecedentes

Phospho-MEK1 (Ser298) se refiere a MEK1, una quinasa de doble especificidad en la cascada MAPK/ERK, fosforilada en la serina 298 por PAK1, lo que mejora la fosforilación mediada por Raf de sus serinas del bucle de activación 218/222 para impulsar la transducción de señales a ERK1/2 para el crecimiento celular, la diferenciación y las respuestas de adhesión. MEK1 presenta un dominio quinasa típico con lóbulos N y C, motivos ricos en prolina y residuos clave, como Asp217 cerca del bucle A, que interactúan con reguladores río arriba, como Arg662 de B-Raf, para facilitar el reposicionamiento del bucle A y la flexibilidad de fosforilación durante la activación. La fosforilación de Ser298 por PAK1 sirve como una convergencia crítica para la señalización de integrinas y factores de crecimiento, localizando phospho-MEK1 en sitios de adhesión periféricos a través de la dependencia de FAK/Src, promoviendo la formación del complejo MEK1-ERK, la activación de MAPK y las respuestas estimuladas por fibronectina como la propagación celular, mientras que los mutantes que carecen de este sitio (S298A) alteran la fosforilación de ERK y la eficiencia de la vía en células NIH/3T3 u otras células. Esta modificación está regulada por la adhesión, es distinta de la fosforilación basal independiente del suero y apoya la transformación o diferenciación sin reactividad cruzada con MEK2.

Información de uso

Aplicación

WB, IP

Dilución

WB	IP
1:1000	1:50

Reactividad

Human, Mouse, Rat

Fuente

Rabbit Monoclonal Antibody

MW

45 kDa

Tampón de almacenamiento

PBS, pH 7.2+50% Glycerol+0.05% BSA+0.01% NaN3

Almacenamiento
(Desde la fecha de recepción)

-20°C (avoid freeze-thaw cycles), 2 years

Métodos experimentales
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.

Métodos experimentales

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.

Referencias

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

Datos de aplicación

WB

Validado por Selleck

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