Cyclin D1 (C-terminal) Antibody [A1D5]

카탈로그 번호 F2524

인쇄

생물학적 설명

특이성

Cyclin D1 (C-terminal) Antibody [A1D5]는 Cyclin D1 단백질의 총 내인성 수준을 검출합니다.
배경

Cell Cycle의 핵심 조절자인 Cyclin D1은 통제되지 않은 세포 증식을 유도하여 암 발생에 중추적인 역할을 합니다. 이 단백질은 11q13 염색체에 위치한 CCND1 유전자에 의해 암호화된 36 kDa 단백질입니다. 구조적으로 Cyclin D1은 RB 단백질 결합 도메인, CDK 또는 그 억제제와 결합하는 영역, 보조활성인자 모집을 위한 LxxLL 모티프, 분해를 위한 PEST 서열, 핵 수출 및 단백질 안정성을 조절하는 중요한 트레오닌 잔기를 포함합니다. Cyclin D1은 골수 줄기세포주에서 유래한 세포를 제외한 대부분의 정상 인간 세포에서 발현됩니다. 그 발현은 성장 인자에 의해 빠르게 유도되어 세포 증식을 제어하는 여러 세포내 신호 경로를 통합하고 조절할 수 있습니다. Cyclin D1 발현, 축적, 유비퀴틴화 및 관련 CDK와의 조립의 조절 이상은 과도한 세포 성장을 유발하며, 이는 Cyclin D1을 유방암, 폐암, 흑색종과 같은 암에서 암 유발 드라이버로 자리매김하게 합니다. 정상 세포에서는 발현이 엄격하게 조절되지만, 암세포는 Cyclin D1 활성을 증폭시키기 위해 다양한 메커니즘을 이용합니다. Cyclin D1은 CDK4 및 CDK6의 알로스테릭 조절자로 작용하여 G1에서 S기로의 전환을 촉진합니다. 활성 Cyclin D1/CDK4 복합체는 핵으로 이동하여 Cyclin E/CDK2와 함께 망막모세포종(RB) 단백질을 인산화합니다. 이 인산화는 RB 매개 E2F 전사 인자 억제를 해제하여 세포 증식에 필수적인 유전자의 전사를 가능하게 합니다. 높은 Cyclin D1 수준은 억제되지 않은 증식과 종양 성장을 유도하며, 이는 암 병인에서 Cyclin D1의 중심 역할을 강조합니다. Cyclin D1은 정규적인 CDK 의존적 기능 외에도 전사 조절자로서 CDK 비의존적 활동을 보입니다. 이는 다양한 전사 인자와 상호작용하여 Cell Cycle 조절 및 증식에 영향을 미칩니다. Cyclin D1은 또한 ERα, AR, 퍼옥시좀 증식자 활성화 수용체 감마(PPARγ), 갑상선 호르몬 수용체 베타(TR-β)와 같은 핵 수용체 및 그 보조 조절자와 상호작용하여 Cell Cycle, 성장 및 분화에 영향을 미칩니다. 또한, 특정 마이크로RNA(miRNA)의 발현을 조절하고 종양-기질 상호작용에 크게 기여하여 다양한 암 특징을 증폭시킵니다. Cyclin D1의 암 시작 및 진행에서의 다면적인 역할은 CDK 의존적 및 CDK 비의존적 세포 기능 조절자로서의 복잡성을 반영합니다. 

사용 정보

응용 WB, IP, IHC, IF 희석
WB IP IHC IF
1:10000 -1: 50000 1:30 1:100 - 1:500 1:50
반응성 Human, Mouse, Rat
출처 Rabbit Monoclonal Antibody MW 34 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, 60 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 1:10000), 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
1238. 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.
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.
 
Permeabilization
1.Add a detergent such as 0.1–0.3% Triton X-100 to the sample and incubate at room temperature for 10–20 minutes.
(Note: This step is only required for intracellular antigens. For antigens expressed on the cell membrane, this step is unnecessary.)
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/33317149/

적용 데이터

WB

Selleck 검증

  • F2524-wb
    Lane 1: A549
    Lane 2: A549 (KO CCND1)
    Lane 3: SH-SY5Y
    Lane 4: Mouse kidney
    Lane 5: Mouse spleen
    Lane 6: Rat heart

IF

Selleck 검증

  • F2524-IF
    Immunofluorescent analysis of MCF-7 cells using F2524 (green, 1:50), Hoechst (blue) and tubulin (Red).