CDKN2A/p16INK4A + CDKN2B/p15INK4B Antibody [C7F9]

카탈로그 번호 F2342

인쇄

생물학적 설명

특이성

CDKN2A/p16INK4A + CDKN2B/p15INK4B Antibody [C7F9]는 CDKN2A/p16INK4A + CDKN2B/p15INK4B 단백질의 내인성 수준을 검출합니다.
배경

CDKN2A는 Cell Cycle의 G1/S기 전환을 조절하는 데 중요한 역할을 하는 사이클린 의존성 키나아제(CDKs)의 구조적으로 관련된 억제제를 암호화하는 유전자군에 속합니다. CDKN2A 유전자는 p16INK4A 단백질을 생성하며, 이 조절자는 CDK4 및 CDK6의 활동을 억제하여 이 상전환을 제어합니다. 인간 염색체 9p21.3에 위치한 CDKN2B 유전자는 p15INK4B를 암호화하며, 이는 CDK4 및 CDK6를 특이적으로 표적으로 하는 또 다른 CDK 억제제입니다. 대안적인 엑손 사용을 통해 p14ARF 및 p16INK4A도 암호화하는 CDKN2A와 함께, 이들 단백질은 p53 및 pRb 경로를 통해 Cell Cycle 진행을 조절합니다. 이 세 가지 CDK 억제제—p15INK4B, p16INK4A 및 p14ARF—는 9p21.3 유전자좌에 탠덤으로 조직되어 중요한 종양 억제 클러스터를 형성합니다. 이 영역은 원발성 세포 불멸화, 전암성 병변 및 다양한 암에서 볼 수 있듯이 초기 종양 형성 동안 자주 삭제되거나 후성적으로 침묵됩니다. 개별 억제제는 프로모터 특이적 메틸화, 시스 작용성 긴 비암호화 안티센스 RNA 또는 발산하는 상류 신호와 같은 별개의 메커니즘을 통해 하향 조절될 수 있습니다. 예를 들어, p15INK4B 발현은 형질전환 성장 인자-베타(TGF-β) 신호 전달에 의해 억제될 수 있습니다. 특히, p15INK4B는 p16INK4A가 없을 때 핵심적인 종양 억제 역할을 수행합니다. 종양 성장을 억제하는 효과는 주요 N-말단 잔기를 통해 CDK4 및 CDK6와 강력하게 상호 작용하고 억제하는 것과 관련이 있습니다. 또한, p15INK4B는 암에서 자주 상향 조절되는 해당 효소인 에놀라제-1을 억제합니다. CDKN2A와 CDKN2B는 모두 잘 확립된 종양 억제 유전자이며 수많은 인간 악성 종양에서 흔히 변형되며, 이는 세포 항상성을 유지하고 종양 진행을 예방하는 데 중요한 역할을 강조합니다. 

사용 정보

응용 WB, IF, FCM 희석
WB IF FCM
1:2000 1:100 - 1:500 1:50 - 1:100
반응성 Human
출처 Rabbit Monoclonal Antibody MW 17 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. ( 0.22 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 1:2000), 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
1246. 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.
 
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.

참조

  • https://pubmed.ncbi.nlm.nih.gov/33824349/
  • https://pubmed.ncbi.nlm.nih.gov/11485924/

적용 데이터

WB

Selleck 검증

  • F2342-wb
    Lane 1: HeLa
    Lane 2: 293

IF

Selleck 검증

  • F2342-IF
    Immunofluorescent analysis of Hela cells using F2342 (green, 1:100), Hoechst (blue) and tubulin (Red).