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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 -- |
팩스: +1-832-582-8590 주문: +1-832-582-8158 |
기술 지원: +1-832-582-8158 Ext:3 이메일에 주문 번호를 기재해 주십시오. 모든 이메일 문의는 영업일 기준 1일 이내에 답변해 드리기 위해 노력하고 있습니다. |
생물학적 설명
| 특이성 | SIRT6 Antibody [H13M15]는 총 SIRT6 단백질의 내인성 수준을 인식합니다. |
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| 배경 | Silent Information Regulator (Sir2) 계열은 니코틴아미드 아데닌 다이뉴클레오타이드(NAD) 의존성 단백질 탈아세틸화효소(클래스 III 히스톤 탈아세틸화효소라고도 함)를 코딩하는 보존된 유전자 그룹입니다. 처음 발견되었고 가장 잘 알려진 구성원은 Saccharomyces cerevisiae의 Sir2로, 교배형 유전자 자리 침묵, 텔로미어 유지, DNA 손상 반응 및 세포 노화에 관여합니다. Sirtuin 6 (SIRT6)는 이 계열의 또 다른 히스톤 탈아세틸화효소로, 다양한 세포 경로에 필수적입니다. SIRT6가 없는 쥐는 자발적인 유전체 불안정성, 조절되지 않은 해당 과정, 종양 형성 증가 및 노화 유사 퇴화의 조기 발병을 보입니다. SIRT6는 핵에 위치하며, DNA 복구를 촉진하고, 대사 유전자 발현을 조절하며, 유전체 안정성을 보장합니다. 이는 종양 억제자이자 장수를 촉진하는 것으로 인정받고 있습니다. SIRT6는 주로 히스톤 및 비히스톤 단백질을 탈아세틸화하여 이러한 과정을 조절하지만, 또한 긴 사슬 탈아실화, ADP-리보실화 및 단백질을 염색질로 모집합니다. SIRT6 기능은 세 가지 주요 클래스로 분류될 수 있습니다: (i) 염색질로 인자 모집, (ii) 히스톤 탈아세틸화를 통한 전사 인자 공동 억제, (iii) 비히스톤 단백질을 탈아세틸화하여 위치 또는 활성 변경. 또한, SIRT6는 DNA 손상 부위에 직접 결합하고 중심 주변 및 텔로미어 DNA를 전반적으로 탈아세틸화하여 비정상적인 전사와 텔로미어 손상을 방지함으로써 선구자 인자 역할을 합니다. |
사용 정보
| 응용 | WB, IP, IF | 희석 |
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| 반응성 | Human, Mouse, Rat, Monkey | ||||||||
| 출처 | Rabbit Monoclonal Antibody | MW | 42, 36 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. ( 0.45 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
712. 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: HCT116
Lane 2: C2C12
Lane 3: COS7
IF
Selleck 검증
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Immunofluorescent analysis of HCT116 cells using F0510 (green, 1:50), Hoechst (blue) and tubulin (Red).