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인용 방법 1. 본문 인용 (재료 및 방법): 2. 주요 자원 표: |
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생물학적 설명
| 특이성 | WRN Antibody [E4K8]는 총 WRN 단백질의 내인성 수준을 검출합니다. |
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| 배경 | RecQ 헬리카제 계열의 구성원인 WRN은 조기 노화 및 암 위험 증가를 특징으로 하는 희귀 상염색체 열성 질환인 베르너 증후군의 원인 유전자입니다. 베르너 증후군 환자의 세포는 손상된 DNA 복구 메커니즘과 관련된 게놈 불안정성을 보입니다. WRN은 독특한 생화학적 활성을 가진 다기능 효소입니다. 이는 ATP-의존성 3'→5' 헬리카제 및 DNA-의존성 ATPase 기능을 나타내며, E. coli DNA 폴리머라제 I의 엑소뉴클레아제 도메인과 유사한 3'→5' 엑소뉴클레아제 활성을 보입니다. WRN의 헬리카제 및 엑소뉴클레아제 기능은 구조적 및 기능적으로 독립적입니다. 예를 들어, WRN1–368과 같은 재조합 N-말단 단편 및 최소 엑소뉴클레아제 도메인 WRN70–240은 엑소뉴클레아제 활성을 유지하지만 헬리카제 기능은 없습니다. WRN의 다양한 역할은 베르너 증후군에서 관찰되는 광범위한 증상과 결합하여 이 효소가 여러 세포 과정에 관여하고 있음을 시사합니다. 이 아이디어를 뒷받침하기 위해, WRN은 증식 세포핵 항원(PCNA), 토포이소머라제 I, 복제 단백질 A(RPA), p53, Ku 복합체 및 DNA 폴리머라제 δ를 포함하여 DNA 대사에 중요한 다양한 단백질과 물리적 및 기능적으로 상호작용하는 것으로 나타났습니다. WRN은 핵 내에서 동적인 국소화를 보이며, 이는 다른 성장 조건에 따라 달라집니다. 여러 세포 유형에서 주로 핵소체에 국소화되며, 그 분포는 DNA 손상 및 세포 주기 단계에 의해 영향을 받습니다. 이러한 상호작용 및 기능적 특성은 게놈 안정성을 유지하고 DNA 손상에 대한 세포 반응을 조절하는 데 WRN의 중요한 역할을 강조합니다. |
사용 정보
| 응용 | WB, IF | 희석 |
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| 반응성 | Human, Mouse, | ||||||
| 출처 | Mouse Monoclonal Antibody | MW | 200 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: 5%. 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, 120 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
1326. 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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| 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.
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참조
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적용 데이터
WB
Selleck 검증
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Lane 1: Hela
Lane 2: Jurkat
Lane 3: Ramos
IF
Selleck 검증
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Immunofluorescent analysis of Hela cells using F1269 (green, 1:1600), Hoechst (blue) and tubulin (Red).