Fiche technique

Description biologique

Spécificité	Phospho-MEK1 (Ser298) Antibody [L22P14] détecte les niveaux endogènes de la protéine MEK1 totale uniquement lorsqu'elle est phosphorylée en Ser298
Contexte	Phospho-MEK1 (Ser298) fait référence à MEK1, une kinase à double spécificité dans la cascade MAPK/ERK, phosphorylée en sérine 298 par PAK1, ce qui améliore la phosphorylation médiatisée par Raf de ses sérines d'activation 218/222 pour entraîner la transduction du signal vers ERK1/2 pour la croissance cellulaire, la différenciation et les réponses d'adhésion. MEK1 présente un domaine kinase typique avec des lobes N et C, des motifs riches en proline et des résidus clés, tels que Asp217 près de la boucle A, qui interagissent avec des régulateurs en amont, comme Arg662 de B-Raf, pour faciliter le repositionnement de la boucle A et la flexibilité de phosphorylation pendant l'activation. La phosphorylation de la Ser298 par PAK1 sert de convergence critique pour la signalisation des intégrines et des facteurs de croissance, localisant la phospho-MEK1 aux sites d'adhésion périphériques via la dépendance FAK/Src, favorisant la formation du complexe MEK1-ERK, l'activation de MAPK et les réponses stimulées par la fibronectine telles que l'étalement cellulaire, tandis que les mutants dépourvus de ce site (S298A) altèrent la phosphorylation d'ERK et l'efficacité de la voie dans les cellules NIH/3T3 ou d'autres cellules. Cette modification est régulée par l'adhésion, distincte de la phosphorylation basale indépendante du sérum, et soutient la transformation ou la différenciation sans réactivité croisée avec MEK2.

Spécificité

Phospho-MEK1 (Ser298) Antibody [L22P14] détecte les niveaux endogènes de la protéine MEK1 totale uniquement lorsqu'elle est phosphorylée en Ser298

Contexte

Phospho-MEK1 (Ser298) fait référence à MEK1, une kinase à double spécificité dans la cascade MAPK/ERK, phosphorylée en sérine 298 par PAK1, ce qui améliore la phosphorylation médiatisée par Raf de ses sérines d'activation 218/222 pour entraîner la transduction du signal vers ERK1/2 pour la croissance cellulaire, la différenciation et les réponses d'adhésion. MEK1 présente un domaine kinase typique avec des lobes N et C, des motifs riches en proline et des résidus clés, tels que Asp217 près de la boucle A, qui interagissent avec des régulateurs en amont, comme Arg662 de B-Raf, pour faciliter le repositionnement de la boucle A et la flexibilité de phosphorylation pendant l'activation. La phosphorylation de la Ser298 par PAK1 sert de convergence critique pour la signalisation des intégrines et des facteurs de croissance, localisant la phospho-MEK1 aux sites d'adhésion périphériques via la dépendance FAK/Src, favorisant la formation du complexe MEK1-ERK, l'activation de MAPK et les réponses stimulées par la fibronectine telles que l'étalement cellulaire, tandis que les mutants dépourvus de ce site (S298A) altèrent la phosphorylation d'ERK et l'efficacité de la voie dans les cellules NIH/3T3 ou d'autres cellules. Cette modification est régulée par l'adhésion, distincte de la phosphorylation basale indépendante du sérum, et soutient la transformation ou la différenciation sans réactivité croisée avec MEK2.

Informations dutilisation

Application

WB, IP

Dilution

WB	IP
1:1000	1:50

Réactivité

Human, Mouse, Rat

Source

Rabbit Monoclonal Antibody

MW

45 kDa

Tampon de stockage

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

Stockage
(À partir de la date de réception)

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

Méthodes expérimentales
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.

Méthodes expérimentales

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.

Références

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

Données dapplication

WB

Validé par Selleck

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