β-Catenin Antibody [M7A19]

카탈로그 번호 F2521

인쇄

생물학적 설명

특이성

β-Catenin Antibody [M7A19]는 총 내인성 β 카테닌 단백질 수준을 검출합니다.
배경

β-카테닌은 접착 연접과 Wnt 신호 전달 경로 내에서 중요한 역할을 하는 다기능 단백질입니다. 세포 내 β-카테닌의 대부분은 접착 연접에 위치하며, 여기서 카드헤린의 세포질 도메인에 결합합니다. 또한 α-카테닌의 동종이량화를 조절하여 액틴 필라멘트 분지 및 번들링에 영향을 미칩니다. 세포질과 핵에서 β-카테닌은 Wnt 신호 전달의 핵심입니다. Wnt 단백질은 배아 발달 및 줄기 세포 유지와 같은 생물학적 과정의 주요 조절자이며, 조절되지 않는 Wnt 신호 전달은 암을 포함한 다양한 질병과 관련이 있습니다. Wnt 신호가 없을 때, 세포질 β-카테닌은 β-카테닌 파괴 복합체에 의해 격리되고 인산화되어 유비퀴틴 매개 프로테아좀 분해를 위해 표시됩니다. Wnt 경로 활성화 시, β-카테닌은 축적되고 핵으로 이동하며 Tcf/LEF 계열 DNA 결합 단백질에 결합하여 캐노니컬 Wnt 반응성 유전자의 전사를 유도합니다. 접착 연접에서 β-카테닌은 E-카드헤린 및 α-카테닌과 상호작용하여 α-카테닌 동종이량체를 통해 액틴 필라멘트와의 연결을 촉진합니다. Wnt 신호가 없을 때, β-카테닌은 파괴 복합체와 결합하여 CK1α 및 GSK-3β에 의해 인산화됩니다. 이 인산화는 β-TrCP 유비퀴틴 리가제에 의한 유비퀴틴화 및 후속 프로테아좀 분해를 유발합니다. Wnt 신호가 있을 때, β-카테닌은 분해를 피하고 핵에 축적되며 Tcf/LEF 계열 단백질 및 기타 전사 인자와 복합체를 형성하여 Wnt 표적 유전자 발현을 활성화합니다. 

사용 정보

응용 WB, IP, IHC, IF, ChIP 희석
WB IP IHC IF CHIP
1:5000-1:10000 1:30 1:500 1:250 1:50
반응성 Human, Mouse, Rat
출처 Rabbit Monoclonal Antibody MW 85 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)，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), 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), 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 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 1:5000), 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
1244. 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. (Exposure time of at least 60s is recommended)
IF
Experimental Protocol:
 
Sample Preparation
1. Adherent Cells: Place a clean, sterile coverslip in a culture dish. Once the cells grow to near confluence as a monolayer, remove the coverslip for further use.
2. Suspension Cells: Seed the cells onto a clean, sterile slide coated with poly-L-lysine.
3. Frozen Sections: Allow the slide to thaw at room temperature. Wash it with pure water or PBS for 2 times, 3 minutes each time.
4. Paraffin Sections: Deparaffinization and rehydration. Wash the slide with pure water or PBS for 3 times, 3 minutes each time. Then perform antigen retrieval.
 
Fixation
1. Fix the cell coverslips/spots or tissue sections at room temperature using a fixative such as 4% paraformaldehyde (4% PFA) for 10-15 minutes.
2. Wash the sample with PBS for 3 times, 3 minutes each time.
 
Blocking
Add blocking solution and incubate at room temperature for at least 1 hour. (Common blocking solutions include: serum from the same source as the secondary antibody, BSA, or goat serum.)
Note: Ensure the sample remains moist during and after the blocking step to prevent drying, which can lead to high background.
 
Immunofluorescence Staining (Day 1)
1. Remove the blocking solution and add the diluted primary antibody.
2. Incubate the sample in a humidified chamber at 4°C overnight.
 
Immunofluorescence Staining (Day 2)
1. Remove the primary antibody and wash with PBST for 3 times, 5 minutes each time.
2. Add the diluted fluorescent secondary antibody and incubate in the dark at 4°C for 1–2 hours.
3. Remove the secondary antibody and wash with PBST for 3 times, 5 minutes each time.
4. Add diluted DAPI and incubate at room temperature in the dark for 5–10 minutes.
5. Wash with PBST for 3 times, 5 minutes each time.
 
Mounting
1. Mount the sample with an anti-fade mounting medium.
2. Allow the slide to dry at room temperature overnight in the dark.
3. Store the slide in a slide storage box at 4°C, protected from light.
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/17881495/#:~:text=It%20functions%20as%20a%20protein,%2D%20and%20C%2Dterminal%20domains.

적용 데이터

WB

Selleck 검증

  • F2521-wb
    Lane 1: HeLa
    Lane 2: MCF7
    Lane 3: MCF7 (KO CTNNB1)

IF

Selleck 검증

  • F2521-IF
    Immunofluorescent analysis of SK-N-SH cells using F2521 (green, 1:250), Hoechst (blue) and tubulin (Red).