Scheda tecnica

Descrizione biologica

Specificità	Phospho-EGF Receptor (Thr669) Antibody [H18D23] rileva i livelli endogeni della proteina EGFR solo quando fosforilata a Thr669.
Contesto	Il recettore del fattore di crescita epidermico (EGFR) è una glicoproteina transmembrana di 170 kDa e un membro della famiglia ErbB di recettori tirosin chinasici (RTK), che include EGFR, ErbB2, ErbB3 ed ErbB4. Questi recettori attivano vie di segnalazione intracellulari come la chinasi proteica attivata da mitogeni (MAPK) e le vie della fosfatidilinositolo 3-chinasi (PI3K)/Akt, che regolano la proliferazione, la differenziazione e l'apoptosi cellulare. La fosforilazione di EGFR a Thr669 avviene in risposta al legame del fattore di crescita epidermico (EGF) al suo dominio extracellulare. Thr669 è un sito regolatore chiave all'interno del dominio citoplasmatico del recettore, situato vicino a due residui di prolina (-Pro-Leu-Thr669-Pro-). La fosforilazione a Thr669 è mediata dalla chinasi regolata dal segnale extracellulare (ERK) e funge da meccanismo di feedback negativo, riducendo l'attività tirosin chinasica di EGFR. Questa regolazione previene una segnalazione eccessiva sopprimendo la fosforilazione tirosinica costitutiva, contribuendo a controllare l'attivazione del recettore e garantendo risposte cellulari equilibrate. Inoltre, la fosforilazione a Thr669 e altri siti (ad es. Ser-1046/7) contribuisce alla segnalazione di stress tramite le vie ERK e p38.

Specificità

Phospho-EGF Receptor (Thr669) Antibody [H18D23] rileva i livelli endogeni della proteina EGFR solo quando fosforilata a Thr669.

Contesto

Il recettore del fattore di crescita epidermico (EGFR) è una glicoproteina transmembrana di 170 kDa e un membro della famiglia ErbB di recettori tirosin chinasici (RTK), che include EGFR, ErbB2, ErbB3 ed ErbB4. Questi recettori attivano vie di segnalazione intracellulari come la chinasi proteica attivata da mitogeni (MAPK) e le vie della fosfatidilinositolo 3-chinasi (PI3K)/Akt, che regolano la proliferazione, la differenziazione e l'apoptosi cellulare. La fosforilazione di EGFR a Thr669 avviene in risposta al legame del fattore di crescita epidermico (EGF) al suo dominio extracellulare. Thr669 è un sito regolatore chiave all'interno del dominio citoplasmatico del recettore, situato vicino a due residui di prolina (-Pro-Leu-Thr669-Pro-). La fosforilazione a Thr669 è mediata dalla chinasi regolata dal segnale extracellulare (ERK) e funge da meccanismo di feedback negativo, riducendo l'attività tirosin chinasica di EGFR. Questa regolazione previene una segnalazione eccessiva sopprimendo la fosforilazione tirosinica costitutiva, contribuendo a controllare l'attivazione del recettore e garantendo risposte cellulari equilibrate. Inoltre, la fosforilazione a Thr669 e altri siti (ad es. Ser-1046/7) contribuisce alla segnalazione di stress tramite le vie ERK e p38.

Informazioni sullutilizzo

Applicazione

WB, IP

Diluizione

WB	IP
1:1000	1:200

Reattività

Human

Fonte

Rabbit Monoclonal Antibody

MW

175 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 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: 5%. 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 946. 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 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: 5%. 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

946. 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/2543683/
https://pubmed.ncbi.nlm.nih.gov/27531070/

Dati di applicazione

WB

Validato da Selleck

Lane 1: A-431 (serum starved overnight), Lane 2: A-431 (serum starved overnight; EGF, 100 ng/ml, 15 min)