Name: 밀집된 핵 환경에서 크로마틴 연구를 위한 다중 표지 단일 분자 위치 현미경 프로토콜 (동영상) | JoVE Journal
Uploaded: 2026-06-05T13:00:00.000+00:00
Duration: 8 min 49 s
Description: 배우세요 다중 표지 단일 분자 위치 현미경 프로토콜을 사용하여 밀집된 핵 환경에서 크로마틴 구조를 정량 분석하는 방법을. BJ Fibroblast, HeLa, MCF 10A 세포에서 나노스케일 공간 조직을 연구할 수 있습니다.

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</ol>

<table><tbody><tr><td>Andor iXon Ultra 888 전자 증폭 CCD&nbsp;</td><td>안도르</td><td>DU-888U3-CSO-#BV&nbsp;</td><td>NA</td></tr><tr><td>소 혈청 알부민</td><td>시그마 올드리치</td><td>A7030</td><td>블로킹 및 워싱 버퍼에 사용됩니다. BSA 기반 차단 버퍼를 4&deg에 1개월 이상 저장하지 마세요; C</td></tr><tr><td>창춘 신산업광전자기술공사, 모델: MGL-FN-532 (532nm) 및 nbsp;</td><td>PSU-H-LED&nbsp;</td><td>https://www.cnilaser.com/MGL-FN-532.htm</td><td>NA</td></tr><tr><td>코히어런트 OBIS 레이저 박스 (405 nm, 488 nm, 532 nm, 552 nm, 637 nm) & nbsp;</td><td>일관성</td><td>1228877&nbsp;</td><td>레이저는 3&ndash와 콜리미네이션을 했습니다; 10 kW/cm&sup3; 샘플 평균 출력, 파장 채널당 최소 10,000프레임 수집; 10&ndash; 30&nbsp; MS&NBSP; 획득 시간.&nbsp;</td></tr><tr><td>DABCO (1,4-디아자비사이클로-(2.2.2)-옥탄)</td><td>시그마</td><td>D27802</td><td>NA</td></tr><tr><td>DBPS (1X)</td><td>써모피셔</td><td>14190-136</td><td>NA</td></tr><tr><td>증류수</td><td>NA</td><td>NA</td><td>증류수는 괜찮아요;</td></tr><tr><td>DTT(디티오트레이톨), 1M</td><td>시그마</td><td>43816</td><td>NA</td></tr><tr><td>8개의 잘 챔버된 커버 유리</td><td>셀비스</td><td>C8-1.5 H-N</td><td>어떤 유리 바닥판이든 가능하지만, 용기에 따라 부피를 조절해야 합니다.</td></tr><tr><td>염소 안티 마우스 AF568</td><td>써모피셔</td><td>A11004</td><td>재고 농도: 2 mg/mL 표기 후 안정성: 2&ndash; 3일</td></tr><tr><td>염소 안티 래빗 AF647</td><td>써모피셔</td><td>A21245</td><td>재고 농도: 2 mg/mL 라벨링 후 안정성: 4&ndash; 5일</td></tr><tr><td>염소 반쥐 A488</td><td>써모피셔</td><td>A11006</td><td>재고 농도: 2 mg/mL 표기 후 안정성: 2&ndash; 3일</td></tr><tr><td>H3K27ac 1차 항체</td><td>써모피셔</td><td>MA5-23516</td><td>재고 농도: 1.0 mg/mL & nbsp;</td></tr><tr><td>H3K9me3 1차 항체 &nbsp;</td><td>앱캠</td><td>AB1769156</td><td>재고 농도: 1.287 mg/mL & nbsp;</td></tr><tr><td>고투명도, 폴리프로필렌 원뿔관 15 mL &nbsp; 그리고 nbsp;</td><td>코닝&nbsp;</td><td>352096</td><td>고정액 및 소금 용액에 사용됨;</td></tr><tr><td>고투명도 폴리프로필렌 원뿔관 50 mL</td><td>코닝</td><td>352070</td><td>40mL 작업 중인 블로킹 및 세척 완충제에 사용</td></tr><tr><td>염산(HCl), 12M</td><td>시그마</td><td>258148</td><td>NA</td></tr><tr><td>완벽한 초점 시스템을 갖춘 니콘 이클립스 Ti-E</td><td>니콘</td><td>TI-DH 611392&nbsp;</td><td>역현미경 &nbsp;</td></tr><tr><td>니콘 SR APO TIRF, 100배 배율, 1.49 NA&nbsp;</td><td>니콘</td><td><a href="https://www.microscope.healthcare.nikon.com/products/optics/cfi-apochromat-tirf-series%C2%A0">https://www.microscope.healthcare.nikon.com/products/optics/cfi-apochromat-tirf-series&nbsp; </a></td><td>NA</td></tr><tr><td>일반 염소 혈청</td><td>앱캠</td><td>AB7481-1002</td><td>첫 번째 라벨이 붙은 후에 사용하세요. 블로킹 및 워싱 버퍼에 있어야 합니다</td></tr><tr><td>파라포름알데히드 16% &nbsp;</td><td>전자현미경 과학</td><td>15710</td><td>고정액에 사용되며, 제작 후 2주 이내에 다 써야 합니다. 빛으로부터 보호하세요 4도 이하; C</td></tr><tr><td>인산염 완충 식염수(PBS) 10X</td><td>암비온</td><td>AM9625</td><td>NA</td></tr><tr><td>피펫 팁 1000 uL</td><td>슈어원</td><td>02-707-404</td><td>어떤 피펫 팁이든 괜찮아요, 단' s는 볼륨에 적합하다</td></tr><tr><td>RNAPII-PS2</td><td>앱캠</td><td>AB252855</td><td>스톡 농도: 0.98 mg/mL</td></tr><tr><td>붕소나트륨</td><td>써모피셔</td><td>S678-10</td><td>매번 신선한 완충 완충제를 사용하세요.&nbsp;</td></tr><tr><td>수산화나트륨(NaOH)</td><td>Thermo Fisher&nbsp;</td><td>A16037.36</td><td>NA</td></tr><tr><td>황산나트륨</td><td>시그마</td><td>S0505</td><td>NA</td></tr><tr><td>트라이튼 X-100 10%</td><td>Thermo Fisher&nbsp;</td><td>28314</td><td>NA</td></tr></tbody></table>

밀집된 핵 환경에서 크로마틴 연구를 위한 다중 표지 단일 분자 위치 현미경 프로토콜

A Multilabel Single Molecule Localization Microscopy Protocol for Investigation of Chromatin in the Dense Nuclear Environment

Super-resolution microscopy has dramatically advanced our ability to interrogate biological structures beyond the diffraction limit, making it ...

a-multilabel-single-molecule-localization-microscopy-protocol-for

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JoVE Journal

Bioengineering

생명공학

단일 분자 현미경은 핵 수송에 새로운 통찰력을 제공 approch.

원자력 교통 단일 분자 이미징

기본 결합 및 고해상도 질량 분석과 크로 마틴 면역 가교으로 ChroP 접근 방식은 히스톤 수정, 변형 및 기능적으로 구별 염색질 영역에서 시너지 효과가 아닌 histonic 단백질의 복합 단백체 구조를 해부 할 수 있습니다.

ChroP 접근은 염색질의 궤적 특정 프로테오믹스 풍경을 해부하는 칩 및 질량 분석을 결합

우리는 면역 염색, 형광 제자리 교잡, 전체 마운트 애기 장대 난자의 양적, 고해상도 이미징 다음 DNA 염색 여기를 효율적이고 신뢰할 수있는 프로토콜을 제공합니다. 이 방법은 성공적 염색질 변형 및 핵 구조를 분석하는데 사용 하였다.

전체 마운트 난자에있는 염색질 수정 및 핵 구조의 정량, 단일 세포 분석을위한 효율적인 방법<em&gt; 애기 장대</em

5-methylcytosine (oxi mCs), 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5 fC)의 산화 형태를 새로 발견 하 고 5-carboxylcytosine (5caC) 독특한 기능 역할을 가진 고유한 DNA 수정 나타낼 수 있습니다. 여기 oxi mCs의 공간 배급, 신호 강도 프로 파일링 및 colocalization의 시각화를 위한 반 정량 워크플로 설명 되어 있습니다.

DNA 수정에 대 한 Immunostaining: 공초점 이미지의 계산 분석

여기, 우리 살아있는 세포의 세포에 단일 막 단백질의 다 색 지역화에 대 한 프로토콜을 제시. Fluorophores 연결할 자체 라벨 단백질 사용 됩니다. 단백질, 세포 기관이 동일한의 다른 막 구획에 있는 ~ 18의 정밀도로 지역화할 수 nm.

살아있는 포유류 세포의 세포에서 단일 막 단백질의 다 색 지역화 현미경

3D 다색 DNA FISH는 3D 보존 핵 내에서 다중 게놈 궤시를 시각화하는 도구를 나타내며, 단일 세포 수준에서 핵 공간 내에서 상호 상호 작용 및 국소화를 명확하게 정의합니다. 여기서, 단계별 프로토콜은 인간 1차 세포의 광범위한 스펙트럼에 대해 기재된다.

인간의 1 차 세포에서 핵 아키텍처를 연구하는 3D 다색 DNA FISH 도구

CUT&amp;RUN 및 그 변이체는 크로마틴에 대한 단백질 점유율을 결정하는 데 사용될 수 있다. 이 프로토콜은 단일 세포 uliCUT&amp;RUN을 사용하여 크로마틴에 대한 단백질 국소화를 결정하는 방법을 설명합니다.

표적 하에서 초저 입력 절단을 이용한 크로마틴에 대한 단일 세포 인자 국소화 및 뉴클레아제를 이용한 방출

본 프로토콜은 단일 분자 국소화 현미경 데이터, 알려진 부피, 게놈 크기 및 세포 주기 단계의 보로노이 테셀레이션을 사용하여 부착 세포 핵 내의 절대 DNA 밀도를 측정하는 방법을 설명합니다.

단일 분자 국소화 현미경을 사용한 세포 핵의 절대 DNA 밀도 매핑

이 기사에서는 단일 분자 상관력 및 형광 현미경을 위해 뉴클레오솜 함유 DNA 테더를 재구성하는 자세한 실험 절차를 제시합니다. 또한 염색질 상호 작용 단백질의 결합 거동을 시각화하고 뉴클레오솜의 물리적 특성 변화를 분석하기 위해 수행할 수 있는 여러 다운스트림 실험에 대해 설명합니다.

In Situ Nucleosome Assembly for Single-Molecule Correlative Force and Fluorescence Microscopy

이 프로토콜은 DNA 고유 형광 스펙트럼의 샘플 준비, 데이터 수집 및 분석에 대한 상세한 작업을 제공합니다.

내재 대비를 이용한 DNA 분광학적 초해상도 영상

이 프로토콜은 최적화된 분리, 고정, 표지를 활용하여 분리된 애기장대 핵의 단일 분자 위치 현미경 영상을 효율적으로 수행하여 크로마틴과 RNA 중합효소 II의 신뢰성 있는 나노 규모 시각화를 달성합니다. 이 접근법은 고해상도 영상을 위한 다른 염색질 관련 단백질이나 히스톤 변형에도 쉽게 적용할 수 있습니다.

애기장대 핵에서 단일 분자 위치 현미경을 위한 간소화된 프로토콜

배우세요 다중 표지 단일 분자 위치 현미경 프로토콜을 사용하여 밀집된 핵 환경에서 크로마틴 구조를 정량 분석하는 방법을. BJ Fibroblast, HeLa, MCF 10A 세포에서 나노스케일 공간 조직을 연구할 수 있습니다.

밀집된 핵 환경에서 크로마틴 연구를 위한 다중 표지 단일 분자 위치 현미경 프로토콜

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