Scheda tecnica

Descrizione biologica

Specificità	Phospho-Smad1 (Ser206) Antibody [D10B24] riconosce i livelli endogeni della proteina SMAD1 solo quando fosforilata a Ser206.
Contesto	Small Body Size (SMA) e Mothers Against Decapentaplegic family 1 (SMAD1), chiamato anche JV4-1, MADH1 o MADR1, si trova sul cromosoma umano 5q4. È stato inizialmente identificato in studi che esploravano i geni coinvolti nella patogenesi del cancro al seno e da allora è stato riconosciuto come un regolatore critico della progressione tumorale. SMAD1 è un mediatore chiave della segnalazione TGF-beta/Smad e svolge un ruolo fondamentale in vari processi biologici, inclusa la crescita cellulare, l'apoptosi, lo sviluppo e le risposte immunitarie. È stato implicato nella progressione di molteplici malignità. SMAD1 transduce segnali da proteine morfogenetiche ossee (BMPs), che regolano diverse attività cellulari come crescita, apoptosi, sviluppo e risposte immunitarie. I ligandi BMP attivano SMAD1 attraverso la fosforilazione da parte delle chinasi del recettore BMP. Una volta fosforilato, SMAD1 forma un complesso con SMAD4, che si trasloca nel nucleo per regolare la trascrizione genica in collaborazione con fattori di trascrizione. È noto che SMAD1 promuove l'invasione cellulare e la metastasi in vari tipi di cancro. Ad esempio, la sovraespressione di SMAD1 migliora la proliferazione delle cellule di cancro allo stomaco in risposta a BMP-7 e supporta la crescita delle cellule di cancro ovarico all'attivazione da BMP-9. Inoltre, le MAP chinasi e le CDK 8 e 9 fosforilano residui nella regione linker di SMAD1, inclusa Ser206. La fosforilazione a Ser206 recluta Smurf1 nella regione linker, portando alla degradazione di SMAD1. È interessante notare che questa fosforilazione migliora anche l'attività trascrizionale di SMAD1 facilitando il reclutamento di YAP nella regione linker.

Specificità

Phospho-Smad1 (Ser206) Antibody [D10B24] riconosce i livelli endogeni della proteina SMAD1 solo quando fosforilata a Ser206.

Contesto

Small Body Size (SMA) e Mothers Against Decapentaplegic family 1 (SMAD1), chiamato anche JV4-1, MADH1 o MADR1, si trova sul cromosoma umano 5q4. È stato inizialmente identificato in studi che esploravano i geni coinvolti nella patogenesi del cancro al seno e da allora è stato riconosciuto come un regolatore critico della progressione tumorale. SMAD1 è un mediatore chiave della segnalazione TGF-beta/Smad e svolge un ruolo fondamentale in vari processi biologici, inclusa la crescita cellulare, l'apoptosi, lo sviluppo e le risposte immunitarie. È stato implicato nella progressione di molteplici malignità. SMAD1 transduce segnali da proteine morfogenetiche ossee (BMPs), che regolano diverse attività cellulari come crescita, apoptosi, sviluppo e risposte immunitarie. I ligandi BMP attivano SMAD1 attraverso la fosforilazione da parte delle chinasi del recettore BMP. Una volta fosforilato, SMAD1 forma un complesso con SMAD4, che si trasloca nel nucleo per regolare la trascrizione genica in collaborazione con fattori di trascrizione. È noto che SMAD1 promuove l'invasione cellulare e la metastasi in vari tipi di cancro. Ad esempio, la sovraespressione di SMAD1 migliora la proliferazione delle cellule di cancro allo stomaco in risposta a BMP-7 e supporta la crescita delle cellule di cancro ovarico all'attivazione da BMP-9. Inoltre, le MAP chinasi e le CDK 8 e 9 fosforilano residui nella regione linker di SMAD1, inclusa Ser206. La fosforilazione a Ser206 recluta Smurf1 nella regione linker, portando alla degradazione di SMAD1. È interessante notare che questa fosforilazione migliora anche l'attività trascrizionale di SMAD1 facilitando il reclutamento di YAP nella regione linker.

Informazioni sullutilizzo

Applicazione

WB, IP

Diluizione

WB	IP
1:1000	1:50

Reattività

Human

Fonte

Rabbit Monoclonal Antibody

MW

60 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 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.

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

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.

Riferimenti

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

Dati di applicazione

WB

Validato da Selleck

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