Fiche technique

Description biologique

Spécificité	VE-cadherin Antibody [C2J1] reconnaît les niveaux endogènes de la protéine VE-cadherin totale.
Contexte	La VE-cadherin, également connue sous le nom de cadhérine endothéliale vasculaire ou CD144, est une protéine transmembranaire critique principalement exprimée dans les cellules endothéliales, où elle joue un rôle vital dans le maintien des jonctions cellule-cellule qui régulent la perméabilité et l'intégrité vasculaire. Membre de la superfamille des cadhérines, la VE-cadherin présente une structure unique composée d'un domaine extracellulaire responsable de la liaison homophilique aux molécules de VE-cadherin adjacentes, d'un seul domaine transmembranaire et d'un domaine intracellulaire qui interagit avec des caténines telles que la β-caténine et la p120-caténine. Cette interaction ancre la VE-cadherin au cytosquelette d'actine, facilitant une forte adhésion entre les cellules endothéliales et formant des jonctions adhérentes essentielles à la stabilité de la barrière endothéliale. La VE-cadherin est cruciale pour l'angiogenèse en régulant la maturation et la stabilisation des structures vasculaires. Elle joue également un rôle significatif dans l'inflammation en modulant le trafic des leucocytes à travers l'endothélium ; pendant les réponses inflammatoires, la phosphorylation de la VE-cadherin peut affaiblir l'adhésion cellule-cellule, permettant aux cellules immunitaires de transmigrer vers les sites de lésion ou d'infection. La régulation de l'activité de la VE-cadherin implique de multiples mécanismes, y compris la phosphorylation par des kinases telles que Src, ce qui influence ses propriétés adhésives et sa perméabilité en réponse à des facteurs de croissance comme le VEGF. Un dérèglement de la VE-cadherin entraîne des cancers et des maladies inflammatoires.

Spécificité

VE-cadherin Antibody [C2J1] reconnaît les niveaux endogènes de la protéine VE-cadherin totale.

Contexte

La VE-cadherin, également connue sous le nom de cadhérine endothéliale vasculaire ou CD144, est une protéine transmembranaire critique principalement exprimée dans les cellules endothéliales, où elle joue un rôle vital dans le maintien des jonctions cellule-cellule qui régulent la perméabilité et l'intégrité vasculaire. Membre de la superfamille des cadhérines, la VE-cadherin présente une structure unique composée d'un domaine extracellulaire responsable de la liaison homophilique aux molécules de VE-cadherin adjacentes, d'un seul domaine transmembranaire et d'un domaine intracellulaire qui interagit avec des caténines telles que la β-caténine et la p120-caténine. Cette interaction ancre la VE-cadherin au cytosquelette d'actine, facilitant une forte adhésion entre les cellules endothéliales et formant des jonctions adhérentes essentielles à la stabilité de la barrière endothéliale. La VE-cadherin est cruciale pour l'angiogenèse en régulant la maturation et la stabilisation des structures vasculaires. Elle joue également un rôle significatif dans l'inflammation en modulant le trafic des leucocytes à travers l'endothélium ; pendant les réponses inflammatoires, la phosphorylation de la VE-cadherin peut affaiblir l'adhésion cellule-cellule, permettant aux cellules immunitaires de transmigrer vers les sites de lésion ou d'infection. La régulation de l'activité de la VE-cadherin implique de multiples mécanismes, y compris la phosphorylation par des kinases telles que Src, ce qui influence ses propriétés adhésives et sa perméabilité en réponse à des facteurs de croissance comme le VEGF. Un dérèglement de la VE-cadherin entraîne des cancers et des maladies inflammatoires.

Informations dutilisation

Application

WB, IP, FCM, ELISA

Dilution

WB	IP	FCM
1:100-1:1000	1:50	1:50

Réactivité

Human, Mouse, Rat, Porcine

Source

Mouse Monoclonal Antibody

MW

130 kDa

Tampon de stockage

PBS, pH 7.2+50% Glycerol+0.05% BSA+0.01% NaN₃

Stockage
(À partir de la date de réception)

-20°C (avoid freeze-thaw cycles), 2 years

Méthodes expérimentales
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: 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 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 1387. 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 60s is recommended)

Méthodes expérimentales

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: 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 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

1387. 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 60s is recommended)

Références

https://pubmed.ncbi.nlm.nih.gov/18162609/
https://pubmed.ncbi.nlm.nih.gov/20708398/

Données dapplication

WB

Validé par Selleck

Lane 1: HUV-EC-C
Lane 2: Human placenta