Fiche technique

Description biologique

Spécificité	Phospho-β-Arrestin 1 (Ser412) Antibody [C17A12] détecte les niveaux endogènes de β-Arrestin 1 uniquement lorsqu'elle est phosphorylée à la sérine 412. L'anticorps ne réagit pas de manière croisée avec β-Arrestin 2.
Contexte	La Phospho-β-Arrestine 1 (Ser412) est essentielle pour contrôler la prolifération et la différenciation des précurseurs de neurones granulaires cérébelleux (CGNP), en particulier dans le cadre de la signalisation Shh. En réponse à Shh, la β-Arrestine 1 subit une phosphorylation au niveau de la Ser412, ce qui favorise son déplacement vers le noyau. Une fois dans le noyau, la β-Arrestine 1 est cruciale pour augmenter la transcription de p27, un inhibiteur de kinase cycline-dépendante qui déclenche la sortie du cycle cellulaire et le début de la différenciation des CGNP. La phosphorylation de la Ser412 améliore l'interaction de la β-Arrestine 1 avec CREB et son recrutement de l'histone acétyltransférase p300, facilitant ainsi l'expression de p27 et affectant la dynamique du cycle cellulaire des CGNP. Ce processus piloté par la phosphorylation crée un mécanisme de rétroaction essentiel au sein de la signalisation Shh, assurant une transition en douceur des CGNP de la prolifération à la différenciation. Cette régulation est cruciale pour assurer un développement cérébelleux normal et a également un impact sur la progression des médulloblastomes induits par Shh.

Spécificité

Phospho-β-Arrestin 1 (Ser412) Antibody [C17A12] détecte les niveaux endogènes de β-Arrestin 1 uniquement lorsqu'elle est phosphorylée à la sérine 412. L'anticorps ne réagit pas de manière croisée avec β-Arrestin 2.

Contexte

La Phospho-β-Arrestine 1 (Ser412) est essentielle pour contrôler la prolifération et la différenciation des précurseurs de neurones granulaires cérébelleux (CGNP), en particulier dans le cadre de la signalisation Shh. En réponse à Shh, la β-Arrestine 1 subit une phosphorylation au niveau de la Ser412, ce qui favorise son déplacement vers le noyau. Une fois dans le noyau, la β-Arrestine 1 est cruciale pour augmenter la transcription de p27, un inhibiteur de kinase cycline-dépendante qui déclenche la sortie du cycle cellulaire et le début de la différenciation des CGNP. La phosphorylation de la Ser412 améliore l'interaction de la β-Arrestine 1 avec CREB et son recrutement de l'histone acétyltransférase p300, facilitant ainsi l'expression de p27 et affectant la dynamique du cycle cellulaire des CGNP. Ce processus piloté par la phosphorylation crée un mécanisme de rétroaction essentiel au sein de la signalisation Shh, assurant une transition en douceur des CGNP de la prolifération à la différenciation. Cette régulation est cruciale pour assurer un développement cérébelleux normal et a également un impact sur la progression des médulloblastomes induits par Shh.

Informations dutilisation

Application

WB, IP

Dilution

WB	IP
1:1000	1:50

Réactivité

Human, Mouse, Rat, Monkey

Source

Mouse Monoclonal Antibody

MW

50 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 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 978. 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 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

978. 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/20935513/

Données dapplication

WB

Validé par Selleck

Lane 1: HEK293
Lane 2: HEK293 (β-arrestin 1 WT)
Lane 3: HEK293 (Ala412 mutant)