Scheda tecnica

Descrizione biologica

Specificità	Phospho-JNK1/2/3 (Tyr185/185/223) Antibody [G4D15] riconosce i livelli endogeni della proteina totale JNK1 (pY185) + JNK2 (pY185) + JNK3 (pY223). Questo anticorpo non riconosce JNK1/2/3 non fosforilata.
Contesto	La proteina chinasi attivata dallo stress/chinasi Jun-ammino-terminale (SAPK/JNK) è notevolmente e prevalentemente attivata da una vasta gamma di stress ambientali e occasionalmente da fattori di crescita e agonisti di GPCR. Similmente ad altre MAPKs, il suo modulo di segnalazione centrale consiste in una MAPKKK, tipicamente MEKK1-MEKK4, o una delle chinasi di lignaggio misto (MLKs), che attivano MKK4/7 attraverso la fosforilazione. Dopo l'attivazione, le MKK fosforilano e attivano ulteriormente la chinasi SAPK/JNK. I segnali di stress vengono trasmessi a questa cascata da piccole GTPasi della famiglia Rho (Rac, Rho, cdc42), con Rac1 e cdc42 che facilitano la stimolazione di MEKKs e MLKs. In alternativa, l'attivazione di MKK4/7 può avvenire indipendentemente dalle GTPasi attraverso la stimolazione di un membro della famiglia della chinasi del centro germinativo (GCK). I geni SAPK/JNK subiscono uno splicing alternativo, producendo numerose isoforme. Quando attiva come dimero, SAPK/JNK può traslocare nel nucleo e modulare la trascrizione influenzando c-Jun, ATF-2 e vari altri fattori di trascrizione.

Specificità

Phospho-JNK1/2/3 (Tyr185/185/223) Antibody [G4D15] riconosce i livelli endogeni della proteina totale JNK1 (pY185) + JNK2 (pY185) + JNK3 (pY223). Questo anticorpo non riconosce JNK1/2/3 non fosforilata.

Contesto

La proteina chinasi attivata dallo stress/chinasi Jun-ammino-terminale (SAPK/JNK) è notevolmente e prevalentemente attivata da una vasta gamma di stress ambientali e occasionalmente da fattori di crescita e agonisti di GPCR. Similmente ad altre MAPKs, il suo modulo di segnalazione centrale consiste in una MAPKKK, tipicamente MEKK1-MEKK4, o una delle chinasi di lignaggio misto (MLKs), che attivano MKK4/7 attraverso la fosforilazione. Dopo l'attivazione, le MKK fosforilano e attivano ulteriormente la chinasi SAPK/JNK. I segnali di stress vengono trasmessi a questa cascata da piccole GTPasi della famiglia Rho (Rac, Rho, cdc42), con Rac1 e cdc42 che facilitano la stimolazione di MEKKs e MLKs. In alternativa, l'attivazione di MKK4/7 può avvenire indipendentemente dalle GTPasi attraverso la stimolazione di un membro della famiglia della chinasi del centro germinativo (GCK). I geni SAPK/JNK subiscono uno splicing alternativo, producendo numerose isoforme. Quando attiva come dimero, SAPK/JNK può traslocare nel nucleo e modulare la trascrizione influenzando c-Jun, ATF-2 e vari altri fattori di trascrizione.

Informazioni sullutilizzo

Applicazione

WB, IP

Diluizione

WB	IP
1:5000	1:70

Reattività

Human, Mouse, Rat

Fonte

Rabbit Monoclonal Antibody

MW

48 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: 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.

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: 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.

Riferimenti

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

Dati di applicazione

WB

Validato da 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)