Scheda tecnica

Descrizione biologica

Specificità	Phospho-MEK1 (Ser298) Antibody [L22P14] rileva i livelli endogeni della proteina MEK1 totale solo quando è fosforilata in Ser298
Contesto	Phospho-MEK1 (Ser298) si riferisce a MEK1, una chinasi a doppia specificità nella cascata MAPK/ERK, fosforilata sulla serina 298 da PAK1, che potenzia la fosforilazione mediata da Raf delle sue serine del loop di attivazione 218/222 per guidare la trasduzione del segnale a ERK1/2 per la crescita cellulare, la differenziazione e le risposte di adesione. MEK1 presenta un tipico dominio chinasico con lobi N e C, motivi ricchi di prolina e residui chiave, come Asp217 vicino all'anello A, che interagiscono con regolatori a monte, come Arg662 di B-Raf, per facilitare il riposizionamento dell'anello A e la flessibilità di fosforilazione durante l'attivazione. La fosforilazione di Ser298 da parte di PAK1 serve come convergenza critica per la segnalazione delle integrine e dei fattori di crescita, localizzando Phospho-MEK1 ai siti di adesione periferici tramite dipendenza da FAK/Src, promuovendo la formazione del complesso MEK1-ERK, l'attivazione di MAPK e le risposte stimolate dalla fibronectina come la diffusione cellulare, mentre i mutanti privi di questo sito (S298A) compromettono la fosforilazione di ERK e l'efficienza della via in NIH/3T3 o altre cellule. Questa modificazione è regolata dall'adesione, distinta dalla fosforilazione basale indipendente dal siero, e supporta la trasformazione o la differenziazione senza reattività crociata con MEK2.

Specificità

Phospho-MEK1 (Ser298) Antibody [L22P14] rileva i livelli endogeni della proteina MEK1 totale solo quando è fosforilata in Ser298

Contesto

Phospho-MEK1 (Ser298) si riferisce a MEK1, una chinasi a doppia specificità nella cascata MAPK/ERK, fosforilata sulla serina 298 da PAK1, che potenzia la fosforilazione mediata da Raf delle sue serine del loop di attivazione 218/222 per guidare la trasduzione del segnale a ERK1/2 per la crescita cellulare, la differenziazione e le risposte di adesione. MEK1 presenta un tipico dominio chinasico con lobi N e C, motivi ricchi di prolina e residui chiave, come Asp217 vicino all'anello A, che interagiscono con regolatori a monte, come Arg662 di B-Raf, per facilitare il riposizionamento dell'anello A e la flessibilità di fosforilazione durante l'attivazione. La fosforilazione di Ser298 da parte di PAK1 serve come convergenza critica per la segnalazione delle integrine e dei fattori di crescita, localizzando Phospho-MEK1 ai siti di adesione periferici tramite dipendenza da FAK/Src, promuovendo la formazione del complesso MEK1-ERK, l'attivazione di MAPK e le risposte stimolate dalla fibronectina come la diffusione cellulare, mentre i mutanti privi di questo sito (S298A) compromettono la fosforilazione di ERK e l'efficienza della via in NIH/3T3 o altre cellule. Questa modificazione è regolata dall'adesione, distinta dalla fosforilazione basale indipendente dal siero, e supporta la trasformazione o la differenziazione senza reattività crociata con MEK2.

Informazioni sullutilizzo

Applicazione

WB, IP

Diluizione

WB	IP
1:1000	1:50

Reattività

Human, Mouse, Rat

Fonte

Rabbit Monoclonal Antibody

MW

45 kDa

Tampone di conservazione

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

Conservazione
(Dalla data di ricevimento)

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

Metodi sperimentali
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.

Metodi sperimentali

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.

Riferimenti

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

Dati di applicazione

WB

Validato da Selleck

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