Phospho-Aurora A/B/C (T288/232/198) Antibody [H12A24]

카탈로그 번호 F0241

인쇄

생물학적 설명

특이성

Phospho-Aurora A/B/C (T288/232/198) Antibody [H12A24]는 Thr288, Thr232 또는 Thr198 중 하나에서 인산화되었을 때만 내인성 Aurora A/B/C 수준을 감지합니다.
배경

Aurora Kinase는 유사분열 세포 분열 조절에 중요한 역할을 하는 세린/트레오닌 키나아제의 새로운 패밀리입니다. 이 패밀리는 Aurora-A, Aurora-B 및 Aurora-C의 세 가지 밀접하게 관련된 구성원으로 구성되며, 이들은 중심체 기능, 양극성 방추체 조립 및 염색체 분리에 관여하는 것으로 알려진 효모 Ipll 및 초파리 Aurora Kinase의 상동체입니다. 세 가지 포유류 Aurora Kinase는 모두 고도로 보존된 촉매 도메인과 짧은 C-말단 도메인 및 다양한 길이의 N-말단 도메인을 공유합니다. Aurora-A는 주로 중심체, 유사분열 방추체 미세소관을 따라, 그리고 유사분열 중인 세포의 세포질에 위치합니다. 단백질 수준은 G1 및 S기 동안 낮지만 세포 주기의 G2/M기 동안 최고조에 달합니다. 촉매 도메인 내 Thr288에서의 Aurora-A 인산화는 키나아제 활성을 향상시키며, 중심체 분리, 성숙, 방추체 조립 및 안정성에 중요합니다. Aurora-B 발현도 G2/M기 동안 최고조에 달하며, 키나아제 활성은 중기부터 유사분열 말기까지 최고 수준에 도달합니다. 전기에 Aurora-B는 염색체와 결합한 다음 후기에 방추체로 재배치됩니다. 미세소관-동원체 부착 및 세포질 분열을 제어하여 염색체 분리를 조절합니다. Aurora-A와 Aurora-B 모두의 발현은 G2/M기 전환 동안 히스톤 H3의 인산화와 긴밀하게 조율되어 유사분열 조절에서 그들의 중요한 역할을 강조합니다. 

사용 정보

응용 WB, IF, FCM 희석
WB IF FCM
1:1000 1:50 1:50
반응성 Human, Mouse, Rat
출처 Rabbit Monoclonal Antibody MW 35, 40, 48 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 1:1000), 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
926. 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.

참조

  • https://pubmed.ncbi.nlm.nih.gov/12884918/
  • https://pubmed.ncbi.nlm.nih.gov/14593731/

적용 데이터

WB

Selleck 검증

  • F0241-wb
    Lane 1: HeLa (hydroxyurea,4 mM,20hrs)
    Lane 2: HeLa (paclitaxel,100 nM,20hrs)

IF

Selleck 검증

  • F0241-IF
    Immunofluorescent analysis of HT1080 cells using F0241 (green, 1:50), Hoechst (blue) and tubulin (Red).