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Descripción biológica

Especificidad	Phospho-(Ser) 14-3-3 Binding Motif Antibody [P9J15] reconoce los niveles endógenos del motivo de unión 14-3-3 solo cuando está fosforilado en el residuo de serina.
Antecedentes	El motivo de unión phospho-(Ser) 14-3-3 es una secuencia clave que se encuentra en las proteínas diana y que interactúa selectivamente con las proteínas 14-3-3 tras la fosforilación en un residuo de serina. Esta fosforilación actúa como un interruptor molecular, activando la unión 14-3-3, y se localiza cerca de regiones funcionales de la proteína diana. Una vez unido, el dímero 14-3-3 estabiliza la proteína, afectando su localización, actividad o estabilidad. Las consecuencias de esta unión son diversas y pueden incluir el secuestro de proteínas en el citoplasma, la inhibición de funciones enzimáticas o el bloqueo de interacciones con otras moléculas. Durante la regulación del ciclo celular, la fosforilación en sitios específicos de proteínas como Cdc25B y DAPK2 provoca la unión 14-3-3, lo que inhibe su función o las dirige al citoplasma. Esta interacción también puede proteger los sitios fosforilados de la desfosforilación, permitiendo que las proteínas diana mantengan sus funciones o permanezcan inactivas según sea necesario. La unión 14-3-3 también desempeña un papel importante en las interacciones proteína-proteína, ya sea protegiendo las proteínas de la degradación o promoviendo el ensamblaje de complejos multiproteicos, que son cruciales para una transducción de señales adecuada.

Especificidad

Phospho-(Ser) 14-3-3 Binding Motif Antibody [P9J15] reconoce los niveles endógenos del motivo de unión 14-3-3 solo cuando está fosforilado en el residuo de serina.

Antecedentes

El motivo de unión phospho-(Ser) 14-3-3 es una secuencia clave que se encuentra en las proteínas diana y que interactúa selectivamente con las proteínas 14-3-3 tras la fosforilación en un residuo de serina. Esta fosforilación actúa como un interruptor molecular, activando la unión 14-3-3, y se localiza cerca de regiones funcionales de la proteína diana. Una vez unido, el dímero 14-3-3 estabiliza la proteína, afectando su localización, actividad o estabilidad. Las consecuencias de esta unión son diversas y pueden incluir el secuestro de proteínas en el citoplasma, la inhibición de funciones enzimáticas o el bloqueo de interacciones con otras moléculas. Durante la regulación del ciclo celular, la fosforilación en sitios específicos de proteínas como Cdc25B y DAPK2 provoca la unión 14-3-3, lo que inhibe su función o las dirige al citoplasma. Esta interacción también puede proteger los sitios fosforilados de la desfosforilación, permitiendo que las proteínas diana mantengan sus funciones o permanezcan inactivas según sea necesario. La unión 14-3-3 también desempeña un papel importante en las interacciones proteína-proteína, ya sea protegiendo las proteínas de la degradación o promoviendo el ensamblaje de complejos multiproteicos, que son cruciales para una transducción de señales adecuada.

Información de uso

Aplicación

WB, IP

Dilución

WB	IP
1:4000	1:20

Reactividad

ALL

Fuente

Rabbit Monoclonal Antibody

MW

29 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)，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), 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), 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: 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, 60 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 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 1:4000), 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 1389. 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)，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), 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), 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: 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, 60 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 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 1:4000), 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

1389. 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/36188227/

Datos de aplicación

WB

Validado por Selleck

Lane 1: A431, Lane 2: A431 (calyculin A, 100nM, 30min)