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인용 방법 1. 본문 인용 (재료 및 방법): 2. 주요 자원 표: |
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무료 전화: (877) 796-6397 -- 미국 및 캐나다 전용 -- |
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생물학적 설명
| 특이성 | Phospho-CDK1/2/3/5 (Tyr15) Antibody [B6L9]는 Y15에서 인산화된 경우에만 내인성 CDK1, CDK2, CDK3 및 CDK5 단백질 수준을 인식합니다. |
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| 배경 | 사이클린 의존성 키나아제(CDK)는 세포 주기 조절, 전사, 통신, 신진대사 및 세포자멸사를 포함한 다양한 필수 세포 과정에서 중추적인 역할을 합니다. 이 키나아제는 세포 분열 동안 정확한 DNA 복제 및 동등한 분리를 보장하기 위해 엄격하게 통제된 경로로 작동합니다. 이러한 과정의 중단은 세포자멸사를 유발할 수 있지만, 교정되지 않고 방치되면 암, 신경퇴행성 질환 및 뇌졸중과 같은 질병을 유발할 수 있습니다. CDK 계열의 20개 구성원이 확인되었으며, 각각 세포 주기, 전사 및 스플라이싱 조절에 기여합니다. 이들 중 이전에 Cdc2라고 불렸던 사이클린 의존성 키나아제 1(CDK1)은 사이클린 B1과 협력하여 G2-에서 유사분열 전환을 유도합니다. CDK1/사이클린 B1 복합체는 G2기 후반에 활성화되어 전중기에서 최대 활동에 도달하고, 방추사 조립 체크포인트가 해결될 때까지 활성 상태를 유지하여 세포가 중기로 진행하도록 합니다. 이 복합체의 활성화에는 CDC25 매개 Thr14 및 Tyr15 억제 부위의 탈인산화가 포함됩니다. 증식하는 세포에서 사이클린 의존성 키나아제 2(CDK2)는 G1/S 및 S/G2 전환의 주요 조절자입니다. CDK2는 사이클린 E와 복합체를 이루어 망막모세포종 단백질(Rb)을 인산화하여 S기 진입을 시작합니다. 또한 CDK2는 다양한 전사 인자의 인산화를 조절하며 세포 분화, 증식, 세포자멸사 및 적응 면역 반응에 중요합니다. 사이클린 의존성 키나아제 5(CDK5)는 다른 CDK와 상당한 서열 유사성을 공유하지만, 주로 유사분열 후 뉴런에서 기능하며, 여기서 신경 구조를 유지하는 데 도움을 줍니다. 사이클린과 결합하는 다른 CDK와 달리 CDK5는 p35 및 p39라는 고유한 서열을 가진 조절 서브유닛에 의해 활성화됩니다. CDK5는 광범위하게 발현되지만, p35 및 p39가 유사분열 후 뉴런에 제한적으로 발현되기 때문에 그 키나아제 활동은 주로 신경계에서 관찰됩니다. |
사용 정보
| 응용 | WB, IP, IHC | 희석 |
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| 반응성 | Human, Rat | ||||||||
| 출처 | Rabbit Monoclonal Antibody | MW | 34 kDa, 65 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, 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 ( 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 1:10000), 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
1249. 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.
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| 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.
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참조
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적용 데이터
WB
Selleck 검증
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Lane 1: HeLa
Lane 2: HeLa (alkaline phosphatase treated)