Scheda tecnica

Descrizione biologica

Specificità	Phospho-Zyxin (Ser142/143) Antibody [L24L6] riconosce i livelli endogeni della proteina zyxin solo quando fosforilata a Ser142 e Ser143.
Contesto	La fosfo-zixina (Ser 142/143) è la forma fosforilata della zixina, una proteina di adesione focale meccanosensibile contenente un dominio LIM e cruciale per l'organizzazione del citoscheletro di actina e la segnalazione cellulare. Espressa in vari tipi cellulari, in particolare cellule endoteliali e muscolari, la sua fosforilazione ai residui di serina 142 e 143—mediata da chinasi come la PKA—migliora la sua interazione con i filamenti di actina e i componenti delle adesioni focali. Strutturalmente, la zixina include una regione ActA ricca di prolina, domini LIM e una regione N-terminale contenente questi siti di fosforilazione, che inducono cambiamenti conformazionali per regolare la sua funzione. La fosfo-zixina svolge ruoli essenziali nell'esocitosi endoteliale, in particolare nella secrezione del fattore di von Willebrand per l'integrità vascolare, e nel rimodellamento del citoscheletro di actina, cruciale per la forma, la motilità e la stabilità meccanica delle cellule. Questi eventi di fosforilazione sono vitali per integrare i segnali extracellulari e modulare le dinamiche del citoscheletro, rendendo la fosfo-zixina fondamentale nella biologia vascolare e nelle risposte cellulari.

Specificità

Phospho-Zyxin (Ser142/143) Antibody [L24L6] riconosce i livelli endogeni della proteina zyxin solo quando fosforilata a Ser142 e Ser143.

Contesto

La fosfo-zixina (Ser 142/143) è la forma fosforilata della zixina, una proteina di adesione focale meccanosensibile contenente un dominio LIM e cruciale per l'organizzazione del citoscheletro di actina e la segnalazione cellulare. Espressa in vari tipi cellulari, in particolare cellule endoteliali e muscolari, la sua fosforilazione ai residui di serina 142 e 143—mediata da chinasi come la PKA—migliora la sua interazione con i filamenti di actina e i componenti delle adesioni focali. Strutturalmente, la zixina include una regione ActA ricca di prolina, domini LIM e una regione N-terminale contenente questi siti di fosforilazione, che inducono cambiamenti conformazionali per regolare la sua funzione. La fosfo-zixina svolge ruoli essenziali nell'esocitosi endoteliale, in particolare nella secrezione del fattore di von Willebrand per l'integrità vascolare, e nel rimodellamento del citoscheletro di actina, cruciale per la forma, la motilità e la stabilità meccanica delle cellule. Questi eventi di fosforilazione sono vitali per integrare i segnali extracellulari e modulare le dinamiche del citoscheletro, rendendo la fosfo-zixina fondamentale nella biologia vascolare e nelle risposte cellulari.

Informazioni sullutilizzo

Applicazione

WB, IP

Diluizione

WB	IP
1:1000	1:200

Reattività

Human, Mouse, Rat, Monkey, Dog

Fonte

Rabbit Monoclonal Antibody

MW

78 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 1:1000), 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 1363. 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. (Exposure time of at least 90s is recommended)

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 1:1000), 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

1363. 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. (Exposure time of at least 90s is recommended)

Riferimenti

https://pubmed.ncbi.nlm.nih.gov/33572997/
https://pubmed.ncbi.nlm.nih.gov/28256511/

Dati di applicazione

WB

Validato da Selleck

Lane 1: A-431
Lane 2: A-431 (λ/CIP treated)
Lane 3: U-2 OS
Lane 4: U-2 OS (λ/CIP treated)