Fiche technique

Description biologique

Spécificité	Phospho-Vimentin (Ser39) Antibody [B14E20] reconnaît les niveaux endogènes de la protéine Vimentin totale uniquement lorsqu'elle est phosphorylée en Ser39.
Contexte	La phospho-vimentine (Ser39) fait référence à la protéine de filament intermédiaire vimentine phosphorylée en sérine 39, une modification qui joue un rôle essentiel dans la régulation de la dynamique des filaments et de l'architecture cellulaire. La vimentine est une protéine de filament intermédiaire de type III principalement exprimée dans les cellules mésenchymateuses, servant de composant majeur du cytosquelette qui fournit une stabilité mécanique, maintient l'intégrité cellulaire et soutient le transport intracellulaire. La vimentine s'assemble de manière hiérarchique : des dimères de coiled-coil solubles s'alignent de manière antiparallèle pour former des tétramères A11, qui s'associent ensuite latéralement et longitudinalement en filaments matures de 11 nm de diamètre. Le domaine de la tête, où se trouve Ser39, est essentiel pour la nucléation de l'assemblage des filaments et sert de point chaud pour la phosphorylation régulatrice. La phosphorylation en Ser39 induit des changements conformationnels qui affaiblissent les interactions des filaments, favorisant le désassemblage ou la réorganisation, un processus crucial pendant la mitose, la migration cellulaire et en réponse au stress mécanique. Ce remodelage dynamique permet à la vimentine de fonctionner comme un échafaudage de signalisation, coordonnant les changements de forme cellulaire, de motilité et de signalisation intracellulaire. Le cycle d'assemblage et de désassemblage réversible, étroitement régulé par des événements de phosphorylation comme en Ser39, permet aux cellules d'adapter rapidement leurs propriétés mécaniques aux demandes physiologiques. La dérégulation de la phosphorylation de la vimentine est impliquée dans la métastase cancéreuse, la fibrose et d'autres pathologies.

Spécificité

Phospho-Vimentin (Ser39) Antibody [B14E20] reconnaît les niveaux endogènes de la protéine Vimentin totale uniquement lorsqu'elle est phosphorylée en Ser39.

Contexte

La phospho-vimentine (Ser39) fait référence à la protéine de filament intermédiaire vimentine phosphorylée en sérine 39, une modification qui joue un rôle essentiel dans la régulation de la dynamique des filaments et de l'architecture cellulaire. La vimentine est une protéine de filament intermédiaire de type III principalement exprimée dans les cellules mésenchymateuses, servant de composant majeur du cytosquelette qui fournit une stabilité mécanique, maintient l'intégrité cellulaire et soutient le transport intracellulaire. La vimentine s'assemble de manière hiérarchique : des dimères de coiled-coil solubles s'alignent de manière antiparallèle pour former des tétramères A11, qui s'associent ensuite latéralement et longitudinalement en filaments matures de 11 nm de diamètre. Le domaine de la tête, où se trouve Ser39, est essentiel pour la nucléation de l'assemblage des filaments et sert de point chaud pour la phosphorylation régulatrice. La phosphorylation en Ser39 induit des changements conformationnels qui affaiblissent les interactions des filaments, favorisant le désassemblage ou la réorganisation, un processus crucial pendant la mitose, la migration cellulaire et en réponse au stress mécanique. Ce remodelage dynamique permet à la vimentine de fonctionner comme un échafaudage de signalisation, coordonnant les changements de forme cellulaire, de motilité et de signalisation intracellulaire. Le cycle d'assemblage et de désassemblage réversible, étroitement régulé par des événements de phosphorylation comme en Ser39, permet aux cellules d'adapter rapidement leurs propriétés mécaniques aux demandes physiologiques. La dérégulation de la phosphorylation de la vimentine est impliquée dans la métastase cancéreuse, la fibrose et d'autres pathologies.

Informations dutilisation

Application

WB

Dilution

WB

1:1000 - 1:200000

Réactivité

Human

Source

Rabbit Monoclonal Antibody

MW

54 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, Phosphatase Inhibitor Cocktail)，and homogenize the tissue at a low temperature. 2. Adherent cell: Aspirate the culture medium and wash the cells with ice-cold PBS twice. Lyse the cells by adding an appropriate volume of RIPA/NP-40 Lysis Buffer (containing Protease Inhibitor Cocktail, Phosphatase Inhibitor Cocktail) and put the sample on ice for 5 min. 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. Lyse the cells by adding an appropriate volume of RIPA/NP-40 Lysis Buffer (containing Protease Inhibitor Cocktail, Phosphatase Inhibitor Cocktail) and put the sample on ice for 5 min. 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 1. 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.

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, Phosphatase Inhibitor Cocktail)，and homogenize the tissue at a low temperature.

2. Adherent cell: Aspirate the culture medium and wash the cells with ice-cold PBS twice. Lyse the cells by adding an appropriate volume of RIPA/NP-40 Lysis Buffer (containing Protease Inhibitor Cocktail, Phosphatase Inhibitor Cocktail) and put the sample on ice for 5 min.

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. Lyse the cells by adding an appropriate volume of RIPA/NP-40 Lysis Buffer (containing Protease Inhibitor Cocktail, Phosphatase Inhibitor Cocktail) and put the sample on ice for 5 min.

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

1. 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.

Références

https://pubmed.ncbi.nlm.nih.gov/38871297/
https://pubmed.ncbi.nlm.nih.gov/38632361/

Données dapplication

WB

Validé par Selleck

Lane 1: Hela, Lane 2: Hela (Calyculin A-treated)