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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 | 5-hydroxymethylcytosine Antibody [B13H23]는 총 5-하이드록시메틸시토신 단백질의 내인성 수준을 검출합니다. |
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| 배경 | 5-하이드록시메틸시토신(5-hmC)은 텐-일레븐 전위(TET) 계열의 다이옥시게나아제(TET1, TET2, TET3)에 의한 5-메틸시토신(5-mC) 산화로 인해 발생하는 독특하고 중요한 후성 유전적 DNA 변형입니다. 이 변형은 시토신 5번 위치에 하이드록시메틸 그룹을 추가하여 염기쌍 형성을 방해하지 않고 DNA의 화학적 환경을 변경하며, 이로 인해 크로마틴 구조와 유전자 조절에 영향을 미칩니다. 5-hmC는 이 하이드록시메틸 그룹의 존재로 5-mC와 다르며, 이는 메틸-CpG 결합 단백질(예: MeCP2)의 결합을 조절하고 하류 효과를 매개하는 특수 판독 단백질을 모집합니다. 5-hmC는 두 가지 역할을 합니다. 하나는 활성 DNA 탈메틸화의 중간체로서, 5-포르밀시토신 및 5-카르복실시토신으로 추가 산화된 후 염기 절제 복구가 이어지며, 다른 하나는 특히 신경 조직에서 유전자 본체, 프로모터 및 인핸서에 풍부한 안정적인 후성 유전적 표지자로서의 역할입니다. 5-hmC의 존재는 개방형 크로마틴 상태를 촉진하여 전사 활성화와 상관 관계가 있으며, 발생 중 및 환경 신호에 반응하여 동적으로 조절됩니다. 5-hmC의 비정상적인 패턴은 종양 발생(예: 교모세포종) 및 레트 증후군과 같은 신경학적 장애와 관련이 있습니다. |
사용 정보
| 응용 | IHC, IF, FCM | 희석 |
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| 반응성 | |||||||||
| 출처 | Rabbit Monoclonal Antibody | MW | |||||||
| 보관 완충액 | PBS, pH 7.2+50% Glycerol+0.05% BSA+0.01% NaN3 | 보관 (수령일로부터) |
-20°C (avoid freeze-thaw cycles), 2 years | ||||||
| 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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| 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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