ID3 Antibody [J14L22]

카탈로그 번호 F2652

인쇄

생물학적 설명

특이성

ID3 Antibody [J14L22]는 총 ID3 단백질의 내인성 수준을 인식합니다.
배경

ID3(Inhibitor of DNA-binding 3)는 헬릭스-루프-헬릭스(HLH) 전사 인자 계열의 구성원으로, 분화, 증식 및 DNA Damage/DNA Repair(DDR)와 같은 핵심 세포 과정을 조절하는 데 중심적인 역할을 합니다. 다른 HLH 단백질과 달리 ID3는 기본적인 DNA 결합 도메인이 없으며, 대신 세포 분화를 촉진하는 전사 인자인 E-단백질에 결합하고 격리하여 기능합니다. 이러한 상호작용은 E-단백질이 분화와 관련된 유전자를 활성화하는 것을 방지하여 전구 세포 또는 미분화 상태를 유지합니다. ID3의 구조는 단백질-단백질 상호작용에 중요하지만 직접적인 DNA 결합에는 중요하지 않은 기본적인 HLH 도메인으로 구성됩니다. DNA Damage/DNA Repair 시 ID3는 DDR의 주요 조절자인 ATM(Ataxia Telangiectasia Mutated) 키나제에 의해 세린 65에서 인산화됩니다. 이 인산화 사건은 DNA Damage/DNA Repair에 관련된 단백질, 특히 MRN 복합체(NBS1, RAD50, MRE11), MDC1 및 RECQL과 같은 이중 가닥 파손(DSB) 수리에 관련된 단백질과의 상호작용을 강화하는 데 필수적입니다. 이러한 상호작용은 상동 재조합(HR)을 통한 수리를 촉진하는 데 중요하며, ID3는 RAD51과 같은 주요 수리 효소의 모집을 촉진하고 DNA 말단 절제를 용이하게 합니다. 또한 전사 조절에도 도움을 주며, E2F1과 같은 전사 인자 및 PRMT5와 같은 염색질 리모델링 인자와의 상호작용을 통해 HR 및 판코니 빈혈(FA) 경로에 관련된 DNA Damage/DNA Repair 유전자의 발현에 영향을 미칩니다. ID3의 손실은 DNA Damage/DNA Repair 손상과 이온화 방사선 및 시스플라틴과 같은 화학 요법제와 같은 DNA 손상 물질에 대한 감수성 증가를 초래합니다. ID3는 암 생물학에서 이중 역할을 하며, 맥락에 따라 종양 억제제와 촉진제 모두로 작용할 수 있습니다. 혈액암 및 결장직장암과 같은 일부 암에서는 ID3가 암 줄기 세포 자가 재생 및 화학 내성을 촉진하는 반면, 다른 암에서는 분화를 촉진하고 통제되지 않은 세포 증식을 방지하여 종양 진행을 억제합니다.

사용 정보

응용 WB, IP, IF, FCM 희석
WB IP IF FCM
1:1000 1:200 1:400 - 1:1600 1:400 - 1:1600
반응성 Human
출처 Rabbit Monoclonal Antibody MW 13 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/Nuclear 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/Nuclear 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/Nuclear 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: 20%. 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 1:1000), 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
1223. 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 300s 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/34718742/
  • https://pubmed.ncbi.nlm.nih.gov/28122577/

적용 데이터

WB

Selleck 검증

  • F2652-wb
    Lane 1: Hela
    Lane 2: Jurkat
    Lane 3: Ramos

IF

Selleck 검증

  • F2652-IF
    Immunofluorescent analysis of Hela cells using F2652 (green, 1:400), Hoechst (blue) and tubulin (Red).