Phospho-Smad1/5/9 (S463/465/467) Antibody [K12G16]

카탈로그 번호 F2875

인쇄

생물학적 설명

특이성

Phospho-Smad1/5/9 (S463/465/467) Antibody [K12G16]는 Ser 463 + Ser 465 + Ser 467에서 인산화되었을 때 전체 Phospho-SMAD1 + SMAD5 + SMAD9의 내인성 수준을 인식합니다.
배경

Small Body Size (SMA) 및 Mothers Against Decapentaplegic family 1 (SMAD1)은 JV4-1, MADH1 또는 MADR1로도 알려져 있으며, 인간 염색체 5q4에 위치한 유전자입니다. 유방암 관련 유전자 연구 중에 처음 확인된 SMAD1은 이후 종양 진행에 있어 중요한 조절자로 인정되었습니다. 이는 TGF-beta/Smad 신호 전달의 핵심 매개체 역할을 하며, 세포 성장, 세포 사멸, 발달 및 면역 반응과 같은 과정에서 필수적인 역할을 합니다. SMAD1은 다양한 악성 종양의 진행에 관여하며, 골 형태 형성 단백질(BMP)의 신호 변환기로 작용하여 증식, 세포 사멸, 발달 및 면역 조절을 포함한 광범위한 세포 기능에 영향을 미칩니다. BMP 리간드는 BMP 수용체 키나아제에 의한 인산화를 통해 SMAD1을 활성화합니다. 일단 인산화되면 SMAD1은 SMAD4와 복합체를 형성하여 핵으로 이동하여 특정 전사 인자와 함께 유전자 전사를 조절합니다. SMAD1은 여러 암 유형에서 세포 침습 및 전이를 촉진하는 것으로 나타났습니다. 예를 들어, SMAD1의 과발현은 BMP-7에 반응하여 위암 세포의 증식을 촉진하고, BMP-9에 의해 활성화될 때 난소암 세포 성장을 향상시킵니다. Ser/Thr 키나아제 수용체의 TGF-beta/Smad 슈퍼패밀리의 일부인 BMP 수용체는 이 신호 전달 캐스케이드에서 중심적인 역할을 합니다. 리간드 결합은 수용체 다중화, 자가인산화 및 활성화를 유발합니다. 활성화된 수용체는 이후 C-말단 영역의 SSXS 모티프 내 특정 세린 잔기(Ser463 및 Ser465)에서 SMAD1을 인산화합니다. 유사한 인산화는 SMAD5 및 SMAD9(SMAD8로도 알려짐)에서도 발생합니다. 이러한 인산화된 SMAD 단백질은 이후 공동 활성제 SMAD4와 이합체를 형성하고 핵으로 이동하여 표적 유전자의 전사를 조절합니다. 

사용 정보

응용 WB, IHC 희석
WB IHC
1:1000-1:10000 1:100 - 1:800
반응성 Human, Mouse, Rat
출처 Rabbit Monoclonal Antibody MW 58 kDa
보관 완충액 PBS, pH 7.2+50% Glycerol+0.05% BSA+0.01% NaN₃
보관
(수령일로부터)		 -20°C (avoid freeze-thaw cycles), 2 years
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
1320. 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.
IHC
Experimental Protocol：
 
Deparaffinization/Rehydration
1. Deparaffinize/hydrate sections:
2. Incubate sections in three washes of xylene for 5 min each.
3. Incubate sections in two washes of 100% ethanol for 10 min each.
4. Incubate sections in two washes of 95% ethanol for 10 min each.
5. Wash sections two times in dH2O for 5 min each.
6.Antigen retrieval: For Citrate: Heat slides in a microwave submersed in 1X citrate unmasking solution until boiling is initiated; continue with 10 min at a sub-boiling temperature (95°-98°C). Cool slides on bench top for 30 min.
 
Staining
1. Wash sections in dH2O three times for 5 min each.
2. Incubate sections in 3% hydrogen peroxide for 10 min.
3. Wash sections in dH2O two times for 5 min each.
4. Wash sections in wash buffer for 5 min.
5. Block each section with 100–400 µl of blocking solution for 1 hr at room temperature.
6. Remove blocking solution and add 100–400 µl primary antibody diluent in to each section. Incubate overnight at 4°C.
7. Remove antibody solution and wash sections with wash buffer three times for 5 min each.
8. Cover section with 1–3 drops HRPas needed. Incubate in a humidified chamber for 30 min at room temperature.
9. Wash sections three times with wash buffer for 5 min each.
10. Add DAB Chromogen Concentrate to DAB Diluent and mix well before use.
11. Apply 100–400 µl DAB to each section and monitor closely. 1–10 min generally provides an acceptable staining intensity.
12. Immerse slides in dH2O.
13. If desired, counterstain sections with hematoxylin.
14. Wash sections in dH2O two times for 5 min each.
15. Dehydrate sections: Incubate sections in 95% ethanol two times for 10 sec each; Repeat in 100% ethanol, incubating sections two times for 10 sec each; Repeat in xylene, incubating sections two times for 10 sec each.
16. Mount sections with coverslips and mounting medium.
 

참조

  • https://pubmed.ncbi.nlm.nih.gov/34134766/
  • https://pubmed.ncbi.nlm.nih.gov/9716398/

적용 데이터

WB

Selleck 검증

  • F2875-wb
    Lane 1: HeLa
    Lane 2: HeLa (BMP-4 treated)
    Lane 3: HeLa (BMP-4 and phosphatase treated)
    Lane 4: Mouse brain
    Lane 5: Rat brain