Fiche technique

Description biologique

Spécificité	Phospho-Smad1 (Ser206) Antibody [D10B24] reconnaît les niveaux endogènes de la protéine SMAD1 uniquement lorsqu'elle est phosphorylée au niveau de la Sérine 206.
Contexte	Small Body Size (SMA) et Mothers Against Decapentaplegic family 1 (SMAD1), également appelé JV4-1, MADH1 ou MADR1, est situé sur le chromosome humain 5q4. Il a été initialement identifié dans des études explorant les gènes impliqués dans la pathogenèse du cancer du sein et a depuis été reconnu comme un régulateur critique de la progression tumorale. SMAD1 est un médiateur clé de la signalisation TGF-beta/Smad et joue un rôle essentiel dans divers processus biologiques, y compris la croissance cellulaire, l'apoptose, le développement et les réponses immunitaires. Il a été impliqué dans la progression de multiples malignités. SMAD1 transduit les signaux des protéines morphogénétiques osseuses (BMP), qui régulent diverses activités cellulaires telles que la croissance, l'apoptose, le développement et les réponses immunitaires. Les ligands BMP activent SMAD1 par phosphorylation par les kinases des récepteurs BMP. Une fois phosphorylé, SMAD1 forme un complexe avec SMAD4, qui se transloque vers le noyau pour réguler la transcription génique en collaboration avec des facteurs de transcription. SMAD1 est connu pour favoriser l'invasion cellulaire et la métastase dans divers types de cancer. Par exemple, la surexpression de SMAD1 améliore la prolifération des cellules cancéreuses de l'estomac en réponse au BMP-7 et soutient la croissance des cellules cancéreuses ovariennes lors de l'activation par le BMP-9. De plus, les MAP kinases et les CDK 8 et 9 phosphorylent des résidus dans la région de liaison de SMAD1, y compris la Sérine 206. La phosphorylation à la Sérine 206 recrute Smurf1 vers la région de liaison, ce qui entraîne la dégradation de SMAD1. Fait intéressant, cette phosphorylation améliore également l'activité transcriptionnelle de SMAD1 en facilitant le recrutement de YAP vers la région de liaison.

Spécificité

Phospho-Smad1 (Ser206) Antibody [D10B24] reconnaît les niveaux endogènes de la protéine SMAD1 uniquement lorsqu'elle est phosphorylée au niveau de la Sérine 206.

Contexte

Small Body Size (SMA) et Mothers Against Decapentaplegic family 1 (SMAD1), également appelé JV4-1, MADH1 ou MADR1, est situé sur le chromosome humain 5q4. Il a été initialement identifié dans des études explorant les gènes impliqués dans la pathogenèse du cancer du sein et a depuis été reconnu comme un régulateur critique de la progression tumorale. SMAD1 est un médiateur clé de la signalisation TGF-beta/Smad et joue un rôle essentiel dans divers processus biologiques, y compris la croissance cellulaire, l'apoptose, le développement et les réponses immunitaires. Il a été impliqué dans la progression de multiples malignités. SMAD1 transduit les signaux des protéines morphogénétiques osseuses (BMP), qui régulent diverses activités cellulaires telles que la croissance, l'apoptose, le développement et les réponses immunitaires. Les ligands BMP activent SMAD1 par phosphorylation par les kinases des récepteurs BMP. Une fois phosphorylé, SMAD1 forme un complexe avec SMAD4, qui se transloque vers le noyau pour réguler la transcription génique en collaboration avec des facteurs de transcription. SMAD1 est connu pour favoriser l'invasion cellulaire et la métastase dans divers types de cancer. Par exemple, la surexpression de SMAD1 améliore la prolifération des cellules cancéreuses de l'estomac en réponse au BMP-7 et soutient la croissance des cellules cancéreuses ovariennes lors de l'activation par le BMP-9. De plus, les MAP kinases et les CDK 8 et 9 phosphorylent des résidus dans la région de liaison de SMAD1, y compris la Sérine 206. La phosphorylation à la Sérine 206 recrute Smurf1 vers la région de liaison, ce qui entraîne la dégradation de SMAD1. Fait intéressant, cette phosphorylation améliore également l'activité transcriptionnelle de SMAD1 en facilitant le recrutement de YAP vers la région de liaison.

Informations dutilisation

Application

WB, IP

Dilution

WB	IP
1:1000	1:50

Réactivité

Human

Source

Rabbit Monoclonal Antibody

MW

60 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 1287. 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

1287. 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/34134766/
https://pubmed.ncbi.nlm.nih.gov/17289590/

Données dapplication

WB

Validé par Selleck

Lane 1: HeLa
Lane 2: HeLa (UV-treated)