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Descripción biológica

Especificidad	Phospho-JNK1/2/3 (Tyr185/185/223) Antibody [G4D15] reconoce los niveles endógenos de la proteína total JNK1 (pY185) + JNK2 (pY185) + JNK3 (pY223). Este anticuerpo no reconoce la JNK1/2/3 no fosforilada.
Antecedentes	La proteína quinasa activada por estrés/quinasa Jun-amino-terminal (SAPK/JNK) se activa de manera notable y predominante por una diversa gama de estreses ambientales y ocasionalmente por factores de crecimiento y agonistas de GPCR. Similar a otras MAPKs, su módulo de señalización central consiste en una MAPKKK, típicamente MEKK1-MEKK4, o una de las quinasas de linaje mixto (MLKs), que activan MKK4/7 a través de la fosforilación. Tras la activación, las MKKs fosforilan y activan aún más la quinasa SAPK/JNK. Las señales de estrés se transmiten a esta cascada por pequeñas GTPasas de la familia Rho (Rac, Rho, cdc42), con Rac1 y cdc42 facilitando la estimulación de MEKKs y MLKs. Alternativamente, la activación de MKK4/7 puede ocurrir independientemente de las GTPasas a través de la estimulación de un miembro de la familia de la quinasa del centro germinal (GCK). Los genes SAPK/JNK sufren un empalme alternativo, produciendo numerosas isoformas. Cuando está activa como dímero, SAPK/JNK puede translocar al núcleo y modular la transcripción influyendo en c-Jun, ATF-2 y varios otros factores de transcripción.

Especificidad

Phospho-JNK1/2/3 (Tyr185/185/223) Antibody [G4D15] reconoce los niveles endógenos de la proteína total JNK1 (pY185) + JNK2 (pY185) + JNK3 (pY223). Este anticuerpo no reconoce la JNK1/2/3 no fosforilada.

Antecedentes

La proteína quinasa activada por estrés/quinasa Jun-amino-terminal (SAPK/JNK) se activa de manera notable y predominante por una diversa gama de estreses ambientales y ocasionalmente por factores de crecimiento y agonistas de GPCR. Similar a otras MAPKs, su módulo de señalización central consiste en una MAPKKK, típicamente MEKK1-MEKK4, o una de las quinasas de linaje mixto (MLKs), que activan MKK4/7 a través de la fosforilación. Tras la activación, las MKKs fosforilan y activan aún más la quinasa SAPK/JNK. Las señales de estrés se transmiten a esta cascada por pequeñas GTPasas de la familia Rho (Rac, Rho, cdc42), con Rac1 y cdc42 facilitando la estimulación de MEKKs y MLKs. Alternativamente, la activación de MKK4/7 puede ocurrir independientemente de las GTPasas a través de la estimulación de un miembro de la familia de la quinasa del centro germinal (GCK). Los genes SAPK/JNK sufren un empalme alternativo, produciendo numerosas isoformas. Cuando está activa como dímero, SAPK/JNK puede translocar al núcleo y modular la transcripción influyendo en c-Jun, ATF-2 y varios otros factores de transcripción.

Información de uso

Aplicación

WB, IP

Dilución

WB	IP
1:5000	1:70

Reactividad

Human, Mouse, Rat

Fuente

Rabbit Monoclonal Antibody

MW

48 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 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: 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 1:5000), 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 722. 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 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: 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 1:5000), 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

722. 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/11274345/
https://pubmed.ncbi.nlm.nih.gov/10557099/

Datos de aplicación

WB

Validado por Selleck

Lane 1: HEK-293
Lane 2: HEK-293 (UV-C, 200J/m2, recovery for 30 min)
Lane 3: NIH/3T3
Lane 4: NIH/3T3 (UV-C, 200J/m2, recovery for 1h)