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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 | Aiolos Antibody [J19G2]는 총 Aiolos 단백질의 내인성 수준을 인식합니다. |
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| 배경 | IKZF3로도 알려진 Aiolos는 아연 손가락 전사 인자 이카로스(Ikaros) 계열의 핵심 구성원으로, 림프구 분화 및 발달에 중요한 역할을 합니다. 여러 아연 손가락 도메인을 통해 DNA에 결합하여 T 및 B 림프구 활동에 필수적인 유전자를 조절합니다. Aiolos는 T 여포 보조 (TFH) 세포의 분화에 특히 중요하며, STAT3와 같은 전사 인자와 협력하여 TFH 세포 발달 및 배 중심 형성의 마스터 조절자인 Bcl-6를 활성화합니다. 또한 사이토카인 수용체와 신호 전달 경로를 조절하여 전염증 반응과 Th1 및 Th17과 같은 대체 면역 프로그램을 조절함으로써 면역 균형을 보장하고, 과도한 면역 활성화 및 자가면역을 방지합니다. Aiolos는 염색질 리모델링을 촉진하여 유전자 접근성을 제어하고, 이카로스 및 헬리오스와 같은 다른 이카로스 계열 구성원과 이합체를 형성하여 유전자 발현을 미세 조정합니다. 분자 스위치 역할을 하여 공동 활성화인자 또는 공동 억제인자를 모집하여 유전자를 활성화하거나 억제할 수 있습니다. B 림프구에서 Aiolos의 주요 역할은 적절한 분화 및 증식을 보장하여 균형 잡힌 면역 반응, 체액 면역 및 건강에 필수적인 전반적인 면역 조절에 필수적입니다. G159R 및 N160S와 같은 Aiolos 돌연변이는 원발성 면역결핍증(PID)과 관련이 있으며, G159R 돌연변이는 B 세포 결핍 및 감염에 대한 감수성 증가와 관련이 있습니다. N160S 돌연변이는 비정상적인 B 세포 표현형 및 T 세포 기능 손상을 초래합니다. |
사용 정보
| 응용 | WB, IP, IHC, FCM, ChIP | 희석 |
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| 반응성 | Human, Mouse | ||||||||||||||
| 출처 | Rabbit Monoclonal Antibody | MW | 50 kDa, 58 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)，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
1390. 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: NK92, Lane 2: Daudi, Lane 3: Raji, Lane 4: A20