Work in the authors' laboratory is supported by a grant from the National Institute of General Medical Sciences (GM41628) and by a grant to the Stowers Institute for Medical Research from the Helen Nelson Medical Research Fund at the Greater Kansas City Community Foundation.

1 ATP에 의존하는 뉴 클레오 리모델링 분석 실험 뉴 클레오 리모델링 활동 ATP-의존성을 측정하기 위해, INO80를 immunopurified 또는 INO80의 서브 콤플렉스는 ATP와 216 bp의, 32 P-표지 된 DNA 단편의 일 단부에 위치하는 단일 뉴 클레오를 포함 mononucleosomal 기판과 함께 배양된다. 반응 생성물은 다음 네이티브 폴리 아크릴 아마이드 겔에서 전기 영동한다. <ol><li> 32 P-표지 된 &#39;, 601&#39;DNA 단편을 생성하기에서 증폭을 pGEM-3Z-601 4 올리고 뉴클레오티드로서 5&#39;-ACAGGATGTATATATCTGACACGTGCCTGG 및 5&#39;-AATACTCAAGCTTGGATGCCTGCAG을 사용하여 최종 위치 (601) 뉴 클레오 솜 위치 서열을 포함하는 216 염기쌍의 DNA 단편, 순방향 및 역방향 프라이머. <ol><li> 표 1에 기재된 바와 같이 100 ㎕의 PCR 반응을 설정한다. </li><li> 다음 프로그램을 사용하여 서열 증폭기에서 PCR 반응을 수행하여 1 분을 96 ° C, followe에서94 ° C, 57 ° C, 30주기 동안 72 ° C에서 60 초에서 30 초에서 45 초만큼 D; 72 ° C에서 7 분으로 끝나는. </li><li> PCR 반응이 완료되면, 클레아없는 스핀 컬럼을 통해 회 반응 생성물을 통과하여 통합되지 않은 뉴클레오타이드를 제거한다. </li><li> PCR 반응은 원하는 ~ 216 염기쌍의 DNA 단편을 생성 된 것을 확인하는 1.2 % 아가 로즈 겔에서 정제 된 PCR 산물의 5 μL를 실행. </li><li> 정제 된 PCR 생성물 20 배량의 5 μL 희석하고 UV 분광 광도계를 이용하여 DNA 농도를 측정한다. 평균 수율은 ~ 40 NG / μL이다. </li><li> 섬광 계수기에서 정제 된 PCR 생성물의 1 μL의 방사능을 측정한다. 노출 당 비용 (CPM) / NG를 계산하여 라벨 효율을 추정한다. 성공적인 라벨링 반응물을 601 DNA 단편 ~ 15,000 CPM / NG을 수득 할 것으로 예상된다. </li></ol></li><li> 용액을 희석 방법 5를 사용하여 표시 601 DNA에 헬라 세포 뉴 클레오을 전송합니다. <ol><li> 2 pmol의 혼합32 P-표지 된 601 DNA 단편 헬라 뉴 클레오 6 μg 1.0 M의 NaCl, 10 mM 트리스 - 염산, pH가 8.0, 1 mM의 EDTA, 0.1 mM의 PMSF 및 1 ㎜를 포함하는 완충액 50 μL에 (같이 제조 5 참조) DTT와 30 분 동안 30 ° C에서 배양한다. </li><li> 순차적 0.8 M, 0.6 M로 혼합물을 희석하고, 10 mM Tris-HCl (pH8.0)을, 1 mM의 EDTA, 0.1 mM의 PMSF의 각각 12.5 μL, 20.8 μL, 41.6 μL, 희석 0.4 M의 NaCl, 및 1 mM의 DTT, 각 희석 한 후 30 ° C에서 30 분 배양과 함께. </li><li> 또한 30 분 동안 배양하여 다음 0.1 % Nonidet P-40, 20 % 글리세롤, 및 200 μg / ㎖ BSA를 함유하는 동일한 완충액 250 μL을 125 μL 첨가함으로써, 0.1 M의 NaCl로 한 다음 0.2 M 및로 혼합물을 희석 마지막이 희석 사이에 30 ° C에서. 최대 3 개월 동안 4 ° C에서 mononucleosome 기판을 저장합니다. </li></ol></li><li> 10 μL의 총 부피에 ATP에 의존하는 뉴 클레오 슬라이딩 반응을 수행합니다. 정말이에요INO80 뉴 클레오 리모델링 활성을위한 최적의 NaCl 농도이기 때문에 ction의 구성 요소. 표 2에 나와있는 것은 ~ 50 mM의 염화나트륨 (미발표 결과), 고려에 함유 된 염화나트륨의 양을 가지고, 50 mm의 각 반응에서의 NaCl의 총 농도를 조정 뉴 클레오 효소의 준비. <ol><li> 분석을 설정하기 시작하기 전에 기본 폴리 아크릴 아마이드 젤 (18 X 16cm)를 캐스팅. 5 %의 아크릴 아미드를 함유 한 겔을 제조 (아크릴 아미드 : 37.5 비스 아크릴 아미드 : 1), 0.5의 TBE (45 mM 트리스 보레이트, 1 mM의 EDTA), 0.01 %과 황산 암모늄 (APS), 및 0.001 % N, N, N&#39;, N&#39;의 -tetramethylethylenediamine (TEMED는). 표 3에 나와있는 재료를 혼합 겔을 실온에서 적어도 2 시간 동안 중합하도록 허용합니다. </li><li> 각각의 반응을 수행하는 한편, 프리 실리콘이 냉장 된 1.5 ml의 마이크로 원심 튜브 ~ 20 nM의 INO80 또는 INO80의 subcomplex에 결합 (EB100 버퍼의 양 (같이 제조 3 참조) <str옹&gt; 충분한 표 4) 4.75 μL의 부피를 수득 하였다. 가루 즉시 드라이 아이스 또는 액체 질소에 남아 INO80 - 함유 분획을 다시 동결. </li><li> &#39;X&#39;의 요소에 의해 확장, 나머지 재료와 함께 마스터 칵테일을 설정합니다 (여기서 반응의 X = 총 세). 단일 10 μL 반응에 필요한 각 성분의 양은 표 5에 열거한다; 레시피가 수행되는 반응의 수에 따라 확장 할 것이다. Pipetman와 튜브를 눌러 또는 피펫 팅 의해 아래로 잘 섞어 벤치 탑의 microcentrifuge에 몇 초 동안 튜브를 회전. </li><li> 단계 1.3.2에서 설정 반응 관 각각에 마스터 칵테일 5.25 μl를 분배. 로 pipetting 아래로 잘 섞는다. 30 ° C 열 블록 또는 물을 욕조에 반응 튜브를 전송하여 반응을 시작하고 2 시간 동안 배양한다. </li><li> 한편, 경쟁 DNA를 포함하는 칵테일 &#39;믹스를 제거하는&#39;준비와 뉴 클레오는 X (반응 + 4 X = 총)의 요인에 의해 확장. 단일 반응 혼합물을 1.5 ㎕의 제거를 준비하기 위해 필요한 각 성분의 양을 표 6에 열거한다; 레시피가 수행되는 분석의 수에 따라 확장 할 것이다. </li><li> 제거 믹스의 1.5 μl를 첨가하여 반응을 종료합니다. 잘 믹스 스핀 다운, 그리고 추가로 30 분 동안 30 ° C에서 배양. </li><li> 한편, 일정한 버퍼 순환을 유지하기 위해 하부 챔버 내부의 자기 교반 막대 버퍼를 실행으로 0.5 배의 TBE를 사용하여, 4 ° C에서 30 분 동안 100 V에서 수직 전기 영동 장치의 기본 폴리 아크릴 아마이드 겔을 미리 실행합니다. </li><li> 샘플을로드 배 TBE, 30 % 글리세롤, 0.25 % 브로 모 페놀 블루, 0.25 % 크실렌 Cyanol FF를 포함 로딩 염료의 2.5 μl를 추가합니다. 간단히 샘플을 회전, 잘 혼합, 및로드 정보를 사용하여 겔에로드합니다. </li><li> 버퍼 순환 4 ° C에서 4.5 시간 동안 200 V에서 젤을 실행합니다.</li><li> 신호를 검출하기 위해, 여과지의 두 시트의 스택 겔 옮긴다. 투명한 플라스틱 포장을 사용하여 상단에있는 겔 필터 종이 랩, 다음 원하는 시간 동안 4 ° C에서 저장 형광체 화면에 노출. </li><li> 동위 원소 촬상 스캐너 시스템의 화면을 검색하고 적합한 소프트웨어를 사용하여 데이터를 분석한다. </li></ol></li></ol> 2 Mononucleosome 결합 분석 mononucleosomes위한 소정 INO80 복합체의 결합 친화도 분석법을 위해, 단계 120에서 생성 mononucleosomal 기판을 이용한 전기 영동 이동성 시프트 분석법 (EMSA)을 수행. <ol><li> 반응은 뉴 클레오 리모델링 분석에 대해 설명하지만 반응에서 ATP 및 제거 믹스를 생략으로 분​​석을 바인딩 믹스 설정; 30 분 동안 30 ° C에서 배양한다. </li><li> 각 반응 혼합물에 로딩 염료의 2.5 μl를 추가하고, 3.5 %의 아크릴 아마이드 (아크릴 아미드가 포함 된 기본 폴리 아크릴 아마이드 겔에 적용 : 세 비스7.5 : 1), 1 % 글리세롤, 0.5 배의 TBE, 0.01 %의 APS 및 0.001 % TEMED. </li><li> 버퍼를 실행으로 0.5 배의 TBE를 사용하여 버퍼 순환 4 ° C에서 2.5 시간 동안 200 V에서 젤을 실행하고 저장 형광체 화면에 노출. </li></ol> 3 DNA- 및 뉴 클레오 의존 ATPase의 분석 실험 20 mM 트리스 - 염산 (pH를 7.5), 60 mM의 염화나트륨, 6.6 mM의 MgCl2를, 0.8 mM의 EDTA, 0.015 % Nonidet P-40, 2.5 % 글리세롤, 0.1 ㎎ / ㎖ BSA, 1을 포함하는 5 ㎕의 반응 혼합물에 ATPase를 세이를 수행 mM의 DTT, 0.1 mM의 PMSF, 2 mM의 ATP [α- 32 P] ATP의 2 μCi (3000 CI / mmol)을 첨가 하였다. INO80 단지의 각 INO80의 복잡하거나 시간 동안 원형 플라스미드 DNA 측정 (5,000 bp의, ~ 30 nm의) 폐쇄 함유 DNA- 또는 뉴 클레오 독립적 ATPase의 하나를 측정하는 세 병렬 반응, 일 포함 EB100 버퍼를 설정 정량 할 DNA- 의존적 ATPase를, 및 뉴 클레오 의존적 ATPase를 측정하는 한 헬러 oligonucleosomes (~ 185 나노 미터)을 포함. 얼음에 모두 반응을 설정합니다. <oL&gt; <li> 각 반응의 경우, 사전 냉장 윤활 1.5 ML의 microcentrifuge 튜브에 2.2 μL의 볼륨을 제공하기에 충분 EB100 버퍼의 양 immunopurified INO80 또는 INO80 서브 콤플렉스의 10 ~ 50 nm의 결합. 가루 즉시 드라이 아이스 또는 액체 질소에있는 INO80 - 함유 분획을 다시 동결. </li><li> 마스터 칵테일을 설정합니다. 단일 반응에 필요한 각 성분의 양을 표 7에 표시됩니다. X는 = INO80 준비의 수를 정량 할 3 (X + 2) 하나의 요인에 의해 확장하여 조리법을 확장 할 수 있습니다. </li><li> &#39;서브 칵테일&#39;포함 버퍼를 준비하려면 만, DNA, 또는 뉴 클레오는 3 개의 별도의 튜브에 마스터 칵테일의 2.5 (X + 2) μl를 분배. 0.3 어느 EB100의 (X + 2) μL 폐쇄 원형 플라스미드 DNA (1.5 μg / μL), 또는 헬라 oligonucleosomes (1.5 μg / μL)을 넣고 잘 섞는다. </li><li> 성에서 설정 한 효소 함유 반응 튜브에 적절한 서브 칵테일의 2.8 μl를 분배EP 3.1. 부드럽게 섞어 아래로 피펫; 거품을 도입하지 마십시오. </li><li> , 반응을 시작하는 30 ° C 열 블록에 반응 튜브를 전송합니다. </li><li> 적어도 1.5 cm 떨어진 하단 가장자리로부터 직선 5, 15, 30, 및 인큐베이션 60 분 후, 셀룰로스 폴리에틸렌 박층 크로마토에 각각 반응 혼합물의 0.5 ㎕를 스팟 (TLC) 플레이트 (20 × 10cm) . 바로 그래서 여러 시점이 하나의 튜브에서 취할 수 있습니다 30 ° C 열 블록에 반응 튜브를 반환합니다. 스포팅 후, 타격 건조기를 사용하여 TLC 판을 건조. </li><li> TLC 플레이트의 하단은 0.5 cm 용액에 침지 될 수 있도록 충분히 0.375 M 인산 칼륨 (pH를 3.5)를 함유하는 유리 챔버 TLC 플레이트를 옮긴다. </li><li> 챔버를 포함하고, 액상의 전면 TLC 플레이트의 상단에 도달 할 때까지 개발한다. 즉시 철저하게 타격 건조기를 사용하여 판을 건조. </li><li> RT에서 저장 형광면에 건조 TLC 판을 노출.동위 원소 촬상 스캐너 시스템의 화면을 스캔 방사성 ATP와 ADP 기판 생성물의 양을 결정한다. </li><li> 가수 분해 ATP의 양을 계산하기 위하여, 다음과 같은 수식을 사용하여 개시, 반응 혼합물 중의 ATP 존재하는 양에 의해 가수 분해 %의 ATP를 곱 pmol의의 ATP 가수 분해 = 10 pmol의의 ATP를 반응 X 개시까지 [ADP / (ATP + ADP)] </li></ol>

<ol>
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D., Thastrom, A., Widom, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spontaneous+access+of+proteins+to+buried+nucleosomal+DNA+target+sites+occurs+via+a+mechanism+that+is+distinct+from+nucleosome+translocation,+Mol.Cell+Biol.">Spontaneous access of proteins to buried nucleosomal DNA target sites occurs via a mechanism that is distinct from nucleosome translocation, Mol.Cell Biol.</a> 22 (20), 7147-7157 (2002).</li><li>Saha, A., Wittmeyer, J., Cairns, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromatin+remodeling+through+directional+DNA+translocation+from+an+internal+nucleosomal+site.">Chromatin remodeling through directional DNA translocation from an internal nucleosomal site.</a> Nature Structural and Molecular Biology. 12 (9), 747-755 (2005).</li><li>Gottschalk, A. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Poly(ADP-ribosyl)ation+directs+recruitment+and+activation+of+an+ATP-dependent+chromatin+remodeler,+Proc.Natl.Acad.Sci.U.S.A.">Poly(ADP-ribosyl)ation directs recruitment and activation of an ATP-dependent chromatin remodeler, Proc.Natl.Acad.Sci.U.S.A.</a> 106 (33), 13770-13774 (2009).</li><li>Clapier, C. R., Cairns, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+ISWI+involves+inhibitory+modules+antagonized+by+nucleosomal+epitopes.">Regulation of ISWI involves inhibitory modules antagonized by nucleosomal epitopes.</a> Nature. 492 (7428), 280-284 (2012).</li><li>Brune, M., Hunter, J. L., Corrie, J. E. T., Direct Webb, M. 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The authors declare that they have no competing financial interests.

, 그리고 리모델링 및 / 또는 ATPase의 효소 오염에 우리가 분석에서 관찰하는 뉴 클레오 리모델링 및 ATPase의 활동 INO80 복합체의 촉매 활성에 따라 보장하기 위해, 우리는 정기적으로 분석 뉴 클레오 리모델링 및 INO80 단지의 촉매 비활성 버전의 ATPase의 활동은 정제 야생형 INO80 동일한 절차를 사용하여 평행. INO80 착체 또는 서브 콤플렉스의 다른 제제의 액티비티를 비교하는 실험의 해석을 복잡하게...

SNF2 가족 염색질 리모델링 단지는 중앙 SNF2 같은 ATPase의 서브 유닛 1, 2를 포함한다. 단일 서브 유니트 효소와 같은 일부 SNF2 형상 ATPases 기능, 다른 큰 멀티 소단위 복합체 촉매 서브 유닛으로서 기능하는 동안. 리모델링 복합체가 그들의 활동에 기여 염색질의 서브 유니트 각각은 리모델링 과정을 해부 생화학 분석을 수행 할 수있는 능력을 필요로하는 분자 메커니즘을 해명. 인간 INO80 착물 및 기타 염색질 개조 효소에 의해 ATP 의존적 뉴 클레오 리모델링은 그 DNA- 및 / 또는 뉴 클레오 의존적 ATPase를 활성화 한 후, 뉴 클레오로 리모델링 효소의 결합을 시작하는 다단계 과정으로 구상 될 수있다 뉴 클레오 솜 DNA, 그리고 뉴 클레오 1,2의 최종 위치 조정에 리모델링 효소의 전위. ATP 의존적 크로 마틴 리모델링 프로세스 (R)의 분자 세부 사항을 이해반응의 각 단계에 크로 마틴 리모델링 복합체의 개별 서브 유닛의 기여의 리모델링의 각 단계에 대한 반응 및 정의의 절개를 equires. 이런 분석은 in vitro에서 정의 된 분자의 기판을 사용하여 뉴 클레오 리모델링 및 기타 활동을 분석 할 수있는 능력을 필요로한다. 이전 조브 프로토콜에서, 우리는 정의 서브 유닛 조성 3 INO80 크로 마틴 리모델링 복합체 및 서브 콤플렉스를 생성하는 데 사용되는 절차를 설명했다. 여기서 우리는, DNA- 및 뉴 클레오-ATPase의 활성화, 이러한 복합체와 관련된 뉴 클레오 리모델링 활동을 결합 뉴 클레오의 정량 분석​​을 가능하게 세 가지 생화학 적 분석을 제시한다.

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biochemical assays for analyzing activities of atp-dependent chromatin remodeling enzymes

Members of the SNF2 family of ATPases often function as components of multi-subunit chromatin remodeling complexes that regulate nucleosome dynamics and DNA accessibility by catalyzing ATP-dependent nucleosome remodeling. Biochemically dissecting the contributions of individual subunits of such complexes to the multi-step ATP-dependent chromatin remodeling reaction requires the use of assays that monitor the production of reaction products and measure the formation of reaction intermediates. This JOVE protocol describes assays that allow one to measure the biochemical activities of chromatin remodeling complexes or subcomplexes containing various combinations of subunits. Chromatin remodeling is measured using an ATP-dependent nucleosome sliding assay, which monitors the movement of a nucleosome on a DNA molecule using an electrophoretic mobility shift assay (EMSA)-based method. Nucleosome binding activity is measured by monitoring the formation of remodeling complex-bound mononucleosomes using a similar EMSA-based method, and DNA- or nucleosome-dependent ATPase activity is assayed using thin layer chromatography (TLC) to measure the rate of conversion of ATP to ADP and phosphate in the presence of either DNA or nucleosomes. Using these assays, one can examine the functions of subunits of a chromatin remodeling complex by comparing the activities of the complete complex to those lacking one or more subunits. The human INO80 chromatin remodeling complex is used as an example; however, the methods described here can be adapted to the study of other chromatin remodeling complexes.

수치는 뉴 클레오 슬라이딩 (그림 1) 및 바인딩 (그림 2) 분석 및 DNA- 또는 뉴 클레오 의존 ATPase의 분석 (그림 3)을 포함 INO80 활동을 특성화하는 데 사용되는 생화학 적 분석의 대표적인 결과를 보여줍니다. 그림 1의 실험은 그대로 INO80 단지의 능력 FLAG-Ies2 또는 FLAG-INO80E을 통해 정제 INO80은 216 BP, 방사성 동위 원소 표지 된 DNA 단...

Watch this Scientific Journal Video about Biochemical Assays for Analyzing Activities of ATP-dependent Chromatin Remodeling Enzymes at JoVE.com

Biochemical Assays for Analyzing Activities of ATP-dependent Chromatin Remodeling Enzymes

Here we describe biochemical assays that can be used to characterize ATP-dependent chromatin remodeling enzymes for their abilities to 1) catalyze ATP-dependent nucleosome sliding, 2) engage with nucleosome substrates, and 3) hydrolyze ATP in a nucleosome- or DNA-dependent manner.

ATP에 의존 염색질 리모델링 효소의 활동을 분석하는 생화학 분석 실험

Members of the SNF2 family of ATPases often function as components of multi-subunit chromatin remodeling complexes that regulate nucleosome dynamics and ...

biochemical-assays-for-analyzing-activities-atp-dependent-chromatin

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JoVE Journal

Biology

14.7K 조회수.  Stowers Institute for Medical Research. Here we describe biochemical assays that can be used to characterize ATP-dependent chromatin remodeling enzymes for their abilities to 1) catalyze ATP-dependent nucleosome sliding, 2) engage with nucleosome substrates, and 3) hydrolyze ATP in a nucleosome- or DNA-dependent manner.

비디오: Biochemical Assays for Analyzing Activities of ATP-dependent Chromatin Remodeling Enzymes

A Chromatin Assay for Human Brain Tissue

Chronic neuropsychiatric illnesses such as schizophrenia, bipolar disease and autism are thought to result from a combination of genetic and environmental factors that might result in epigenetic alterations of gene expression and other molecular pathology. Traditionally, however, expression studies in postmortem brain were confined to quantification of mRNA or protein. The limitations encountered in postmortem brain research   such as variabilities in autolysis time and tissue integrities   are also likely to impact any studies of higher order chromatin structures. However, the nucleosomal organization of genomic DNA   including DNA:core histone binding - appears to be largely preserved in representative samples provided by various brain banks. Therefore, it is possible to study the methylation pattern and other covalent modifications of the core histones at defined genomic loci in postmortem brain. Here, we present a simplified native chromatin immunoprecipitation (NChIP) protocol for frozen (never-fixed) human brain specimens. Starting with micrococcal nuclease digestion of brain homogenates, NChIP followed by qPCR can be completed within three days. The methodology presented here should be useful to elucidate epigenetic mechanisms of gene expression in normal and diseased human brain.

Until recently, expression studies on human brain were limited to quantification of RNA or protein. With the chromatin immunoprecipitation techniques described in this paper, it will be possible to map histone methylation and other epigenetic regulators of gene expression in postmortem brain.

인간 두뇌 조직에 대한 염색질 어세이

Chronic neuropsychiatric illnesses such as schizophrenia, bipolar disease and autism are thought to result from a combination of genetic and environmental ...

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생물학

Zebrafish Brain Ventricle Injection

Proper brain ventricle formation during embryonic brain development is required for normal brain function.  Brain ventricles are the highly conserved cavities within the brain that are filled with cerebrospinal fluid.  In zebrafish, after neural tube formation, the neuroepithelium undergoes a series of constrictions and folds while it fills with fluid resulting in brain ventricle formation.  In order to understand the process of ventricle formation, and the neuroepithelial shape changes that occur at the same time, we needed a way to visualize the ventricle space in comparison to the brain tissue.  However, the nature of transparent zebrafish embryos makes it difficult to differentiate the tissue from the ventricle space.  Therefore, we developed a brain ventricle injection technique where the ventricle space is filled with a fluorescent dye and imaged by brightfield and fluorescent microscopy.  The brightfield and the fluorescent images are then processed and superimposed in Photoshop.  This technique allows for visualization of the ventricle space with the fluorescent dye, in comparison to the shape of the neuroepithelium in the brightfield image.  Brain ventricle injection in zebrafish can be employed from 18 hours post fertilization through early larval stages.  We have used this technique extensively in our studies of brain ventricle formation and morphogenesis as well as in characterizing brain morphogenesis mutants (1-3).

After neural tube formation, the neuroepithelium constricts and folds while the tube fills with embryonic cerebrospinal fluid (eCSF) to form the embryonic brain ventricles. We developed this ventricle injection technique to better visualize the fluid filled space in contrast to the neuroepithelial shape in a live embryo.

Zebrafish의 두뇌 뇌실 주입

Proper brain ventricle formation during embryonic brain development is required for normal brain function.  Brain ventricles are the highly conserved ...

Photobleaching Assays (FRAP &amp; FLIP) to Measure Chromatin Protein Dynamics in Living Embryonic Stem Cells

Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Loss In Photobleaching (FLIP) enable the study of protein dynamics in living cells with good spatial and temporal resolution. Here we describe how to perform FRAP and FLIP assays of chromatin proteins, including H1 and HP1, in mouse embryonic stem (ES) cells. In a FRAP experiment, cells are transfected, either transiently or stably, with a protein of interest fused with the green fluorescent protein (GFP) or derivatives thereof (YFP, CFP, Cherry, etc.). In the transfected, fluorescing cells, an intense focused laser beam bleaches a relatively small region of interest (ROI). The laser wavelength is selected according to the fluorescent protein used for fusion. The laser light irreversibly bleaches the fluorescent signal of molecules in the ROI and, immediately following bleaching, the recovery of the fluorescent signal in the bleached area - mediated by the replacement of the bleached molecules with the unbleached molecules - is monitored using time lapse imaging. The generated fluorescence recovery curves provide information on the protein's mobility. If the fluorescent molecules are immobile, no fluorescence recovery will be observed. In a complementary approach, Fluorescence Loss in Photobleaching (FLIP), the laser beam bleaches the same spot repeatedly and the signal intensity is measured elsewhere in the fluorescing cell. FLIP experiments therefore measure signal decay rather than fluorescence recovery and are useful to determine protein mobility as well as protein shuttling between cellular compartments. Transient binding is a common property of chromatin-associated proteins. Although the major fraction of each chromatin protein is bound to chromatin at any given moment at steady state, the binding is transient and most chromatin proteins have a high turnover on chromatin, with a residence time in the order of seconds. These properties are crucial for generating high plasticity in genome expression1. Photobleaching experiments are therefore particularly useful to determine chromatin plasticity using GFP-fusion versions of chromatin structural proteins, especially in ES cells, where the dynamic exchange of chromatin proteins (including heterochromatin protein 1 (HP1), linker histone H1 and core histones) is higher than in differentiated cells2,3.

Photobleaching Assays FRAP & FLIP to Measure Chromatin Protein Dynamics in Living Embryonic Stem Cells

We describe photobleaching methods including Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Loss In Photobleaching (FLIP) to monitor chromatin protein dynamics in embryonic stem (ES) cells. Chromatin protein dynamics, which is considered to be one of the means to study chromatin plasticity, is enhanced in pluripotent cells.

배아 줄기 세포 생활에서 염색질 단백질 역학을 측정하는 Assays (FRAP 및 플립)를 Photobleaching

Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Loss In Photobleaching (FLIP) enable the study of protein dynamics in living cells with ...

Two Types of Assays for Detecting Frog Sperm Chemoattraction

Sperm chemoattraction in invertebrates can be sufficiently robust that one can place a pipette containing the attractive peptide into a sperm suspension and microscopically visualize sperm accumulation around the pipette1. Sperm chemoattraction in vertebrates such as frogs, rodents and humans is more difficult to detect and requires quantitative assays. Such assays are of two major types - assays that quantitate sperm movement to a source of chemoattractant, so-called sperm accumulation assays, and assays that actually track the swimming trajectories of individual sperm.
Sperm accumulation assays are relatively rapid allowing tens or hundreds of assays to be done in a single day, thereby allowing dose response curves and time courses to be carried out relatively rapidly. These types of assays have been used extensively to characterize many well established chemoattraction systems - for example, neutrophil chemotaxis to bacterial peptides and sperm chemotaxis to follicular fluid. Sperm tracking assays can be more labor intensive but offer additional data on how chemoattractancts actually alter the swimming paths that sperm take. This type of assay is needed to demonstrate the orientation of sperm movement relative to the chemoattrractant gradient axis and to visualize characteristic turns or changes in orientation that bring the sperm closer to the egg.
Here we describe methods used for each of these two types of assays. The sperm accumulation assay utilized is called a "two-chamber" assay. Amphibian sperm are placed in a tissue culture plate insert with a polycarbonate filter floor having 12 &mu;m diameter pores. Inserts with sperm are placed into tissue culture plate wells containing buffer and a chemoatttractant carefully pipetted into the bottom well where the floor meets the wall (see Fig. 1). After incubation, the top insert containing the sperm reservoir is carefully removed, and sperm in the bottom chamber that have passed through the membrane are removed, pelleted and then counted by hemocytometer or flow cytometer.
The sperm tracking assay utilizes a Zigmond chamber originally developed for observing neutrophil chemotaxis and modified for observation of sperm by Giojalas and coworkers2,3. The chamber consists of a thick glass slide into which two vertical troughs have been machined. These are separated by a 1 mm wide observation platform. After application of a cover glass, sperm are loaded into one trough, the chemoattractant agent into the other and movement of individual sperm visualized by video microscopy. Video footage is then analyzed using software to identify two-dimensional cell movements in the x-y plane as a function of time (xyt data sets) that form the trajectory of each sperm.

Eggs and the extracellular coatings around eggs frequently release peptides, proteins and small molecules that communicate with sperm to guide them to the egg thereby promoting fertilization. Using frog sperm we describe and compare two classes of assays used to detect sperm chemoattraction &#x2013; sperm accumulation assays and sperm tracking assays.

감지 개구리 정자 Chemoattraction에 대한 Assays 두 종류의

Sperm chemoattraction in invertebrates can be sufficiently robust that one can place a pipette containing the attractive peptide into a sperm suspension ...

Chromatin Interaction Analysis with Paired-End Tag Sequencing (ChIA-PET) for Mapping Chromatin Interactions and Understanding Transcription Regulation

Genomes are organized into three-dimensional structures, adopting higher-order conformations inside the micron-sized nuclear spaces 7, 2, 12. Such architectures are not random and involve interactions between gene promoters and regulatory elements 13. The binding of transcription factors to specific regulatory sequences brings about a network of transcription regulation and coordination 1, 14. 
Chromatin Interaction Analysis by Paired-End Tag Sequencing (ChIA-PET) was developed to identify these higher-order chromatin structures 5,6. Cells are fixed and interacting loci are captured by covalent DNA-protein cross-links. To minimize non-specific noise and reduce complexity, as well as to increase the specificity of the chromatin interaction analysis, chromatin immunoprecipitation (ChIP) is used against specific protein factors to enrich chromatin fragments of interest before proximity ligation. Ligation involving half-linkers subsequently forms covalent links between pairs of DNA fragments tethered together within individual chromatin complexes. The flanking MmeI restriction enzyme sites in the half-linkers allow extraction of paired end tag-linker-tag constructs (PETs) upon MmeI digestion. As the half-linkers are biotinylated, these PET constructs are purified using streptavidin-magnetic beads. The purified PETs are ligated with next-generation sequencing adaptors and a catalog of interacting fragments is generated via next-generation sequencers such as the Illumina Genome Analyzer. Mapping and bioinformatics analysis is then performed to identify ChIP-enriched binding sites and ChIP-enriched chromatin interactions 8. 
We have produced a video to demonstrate critical aspects of the ChIA-PET protocol, especially the preparation of ChIP as the quality of ChIP plays a major role in the outcome of a ChIA-PET library. As the protocols are very long, only the critical steps are shown in the video.

Chromatin Interaction Analysis with Paired-End Tag Sequencing ChIA-PET for Mapping Chromatin Interactions and Understanding Transcription Regulation

Chromatin Interaction Analysis by Paired-End Tag Sequencing (ChIA-PET) is a method for de novo detection of chromatin interactions, for better understanding of transcriptional control.

염색질 상호 작용을 매핑 및 전사 규정을 이해하는 데 이점 엔드 태그 장면 (ChIA-PET)과 염색질의 상호 작용 분석

Genomes are organized into three-dimensional structures, adopting higher-order conformations inside the micron-sized nuclear spaces 7, 2, 12. Such ...

Blood Collection for Biochemical Analysis in Adult Zebrafish

The zebrafish has been used as an animal model for studies of several human diseases. It can serve as a powerful preclinical platform for studies of molecular events and therapeutic strategies as well as for evaluating the physiological mechanisms of some pathologies1. 
There are relatively few publications related to adult zebrafish physiology of organs and systems2, which may lead researchers to infer that the basic techniques needed to allow the exploration of zebrafish systems are lacking3. Hematologic biochemical values of zebrafish were first reported in 2003 by Murtha and colleagues4 who employed a blood collection technique first described by Jagadeeswaran and colleagues in 1999. Briefly, blood was collected via a micropipette tip through a lateral incision, approximately 0.3 cm in length, in the region of the dorsal aorta5. Because of the minute dimensions involved, this is a high-precision technique requiring a highly skilled practitioner. The same technique was used by the same group in another publication in that same year6. In 2010, Eames and colleagues assessed whole blood glucose levels in zebrafish7. They gained access to the blood by performing decapitations with scissors and then inserting a heparinized microcapillary collection tube into the pectoral articulation. They mention difficulties with hemolysis that were solved with an appropriate storage temperature based on the work Kilpatrick et al.8. When attempting to use Jagadeeswaran's technique in our laboratory, we found that it was difficult to make the incision in precisely the right place as not to allow a significant amount of blood to be lost before collection could be started. 
Recently, Gupta et al.9 described how to dissect adult zebrafish organs, Kinkle et al.10 described how to perform intraperitoneal injections, and Pugach et al.11 described how to perform retro-orbital injections. However, more work is needed to more fully explore basic techniques for research in zebrafish.
The small size of zebrafish presents challenges for researchers using it as an experimental model. Furthermore, given this smallness of scale, it is important that simple techniques are developed to enable researchers to explore the advantages of the zebrafish model.

This paper presents a technique for collection of blood from the dorsal aorta of Zebrafish. It also provides instructions for obtaining serum for use in biochemical analyses, such as tests for determining cholesterol and triglyceride levels.

성인 Zebrafish의 생물 화학 분석을위한 혈액 수집

The zebrafish has been used as an animal model for studies of several human diseases. It can serve as a powerful preclinical platform for studies of ...

Extraction of Tissue Antigens for Functional Assays

Many of the antigen targets of adaptive immune response, recognized by B and T cells, have not been defined 1. This is particularly true in autoimmune diseases and cancer2. Our aim is to investigate the antigens recognized by human T cells in the autoimmune disease type 1 diabetes 1,3,4,5. To analyze human T-cell responses against tissue where the antigens recognized by T cells are not identified we developed a method to extract protein antigens from human tissue in a format that is compatible with functional assays 6. Previously, T-cell responses to unpurified tissue extracts could not be measured because the extraction methods yield a lysate that contained detergents that were toxic to human peripheral blood mononuclear cells. Here we describe a protocol for extracting proteins from human tissues in a format that is not toxic to human T cells. The tissue is homogenized in a mixture of butan-1-ol, acetonitrile and water (BAW). The protein concentration in the tissue extract is measured and a known mass of protein is aliquoted into tubes. After extraction, the organic solvents are removed by lyophilization. Lyophilized tissue extracts can be stored until required. For use in assays of immune function, a suspension of immune cells, in appropriate culture media, can be added directly to the lyophilized extract. Cytokine production and proliferation by PBMC, in response to extracts prepared using this method, were readily measured. Hence, our method allows the rapid preparation of human tissue lysates that can be used as a source of antigens in the analysis of T-cell responses. We suggest that this method will facilitate the analysis of adaptive immune responses to tissues in transplantation, cancer and autoimmunity.

A simple protocol for preparing extracts of human tissue to be used as a source of antigens in functional T-cell assays is described. This method allows T-cell responses to tissue-derived antigens to be measured in vitro.

기능 Assays을위한 조직 항원의 추출

Many of the antigen targets of adaptive immune response, recognized by B and T cells, have not been defined 1. This is particularly true in autoimmune ...

Biochemical and High Throughput Microscopic Assessment of Fat Mass in Caenorhabditis Elegans

The nematode C. elegans has emerged as an important model for the study of conserved genetic pathways regulating fat metabolism as it relates to human obesity and its associated pathologies. Several previous methodologies developed for the visualization of C. elegans triglyceride-rich fat stores have proven to be erroneous, highlighting cellular compartments other than lipid droplets. Other methods require specialized equipment, are time-consuming, or yield inconsistent results. We introduce a rapid, reproducible, fixative-based Nile red staining method for the accurate and rapid detection of neutral lipid droplets in C. elegans. A short fixation step in 40% isopropanol makes animals completely permeable to Nile red, which is then used to stain animals. Spectral properties of this lipophilic dye allow it to strongly and selectively fluoresce in the yellow-green spectrum only when in a lipid-rich environment, but not in more polar environments. Thus, lipid droplets can be visualized on a fluorescent microscope equipped with simple GFP imaging capability after only a brief Nile red staining step in isopropanol. The speed, affordability, and reproducibility of this protocol make it ideally suited for high throughput screens. We also demonstrate a paired method for the biochemical determination of triglycerides and phospholipids using gas chromatography mass-spectrometry. This more rigorous protocol should be used as confirmation of results obtained from the Nile red microscopic lipid determination. We anticipate that these techniques will become new standards in the field of C. elegans metabolic research.

Biochemical and High Throughput Microscopic Assessment of Fat Mass in Caenorhabditis Elegans

We present robust biochemical and microscopic methods for studying Caenorhabditis elegans lipid stores. A rapid, simple, fixing-staining procedure for fluorescent lipid droplet imaging leverages the spectral properties of the lipophilic dye Nile red. We then present biochemical measurement of triglycerides and phospholipids using solid phase extraction and gas chromatography-mass spectrometry.

에서 지방 질량의 생화학 및 높은 처리량 미세 평가<em&gt; Caenorhabditis Elegans</em

The nematode C. elegans has emerged as an important  model for the study of conserved genetic pathways regulating fat metabolism as  it relates to human ...

Biochemical Titration of Glycogen In vitro

Glycogen is the main energetic polymer of glucose in vertebrate animals and plays a crucial role in whole body metabolism as well as in cellular metabolism. Many methods to detect glycogen already exist but only a few are quantitative. We describe here a method using the Abcam Glycogen assay kit, which is based on specific degradation of glycogen to glucose by glucoamylase. Glucose is then specifically oxidized to a product that reacts with the OxiRed probe to produce fluorescence. Titration is accurate, sensitive and can be achieved on cell extracts or tissue sections. However, in contrast to other techniques, it does not give information about the distribution of glycogen in the cell. As an example of this technique, we describe here the titration of glycogen in two cell lines, Chinese hamster lung fibroblast CCL39 and human colon carcinoma LS174, incubated in normoxia (21% O2) versus hypoxia (1% O2). We hypothesized that hypoxia is a signal that prepares cells to synthesize and store glycogen in order to survive1.

Biochemical Titration of Glycogen In vitro

We describe here an accurate, reproducible and convenient biochemical method for titration of glycogen in vitro. This technique uses the Abcam Glycogen assay kit and is based on successive hydrolysis of glycogen to glucose and glucose titration by fluorescence.

글리코겐의 생화학 적정<em&gt; 체외</em

Glycogen is the main energetic polymer of glucose in vertebrate animals and plays a crucial role in whole body metabolism as well as in cellular ...

Viability Assays for Cells in Culture

Manual cell counts on a microscope are a sensitive means of assessing cellular viability but are time-consuming and therefore expensive. Computerized viability assays are expensive in terms of equipment but can be faster and more objective than manual cell counts. The present report describes the use of three such viability assays. Two of these assays are infrared and one is luminescent. Both infrared assays rely on a 16&#160;bit Odyssey Imager. One infrared assay uses the DRAQ5 stain for nuclei combined with the Sapphire stain for cytosol and is visualized in the 700 nm channel. The other infrared assay, an In-Cell Western, uses antibodies against cytoskeletal proteins (&#945;-tubulin or microtubule associated protein 2) and labels them in the 800 nm channel. The third viability assay is a commonly used luminescent assay for ATP, but we use a quarter of the recommended volume to save on cost. These measurements are all linear and correlate with the number of cells plated, but vary in sensitivity. All three assays circumvent time-consuming microscopy and sample the entire well, thereby reducing sampling error. Finally, all of the assays can easily be completed within one day of the end of the experiment, allowing greater numbers of experiments to be performed within short timeframes. However, they all rely on the assumption that cell numbers remain in proportion to signal strength after treatments, an assumption that is sometimes not met, especially for cellular ATP. Furthermore, if cells increase or decrease in size after treatment, this might affect signal strength without affecting cell number. We conclude that all viability assays, including manual counts, suffer from a number of caveats, but that computerized viability assays are well worth the initial investment. Using all three assays together yields a comprehensive view of cellular structure and function.

Therapeutic compounds are often first examined in vitro with viability assays. Blind cell counts by a human observer can be highly sensitive to small changes in cell number but do not assess function. Computerized viability assays, as described here, can assess both structure and function in an objective manner.

문화의 세포에 대한 생존 분석 실험

Manual cell counts on a microscope are a sensitive means of assessing cellular viability but are time-consuming and therefore expensive. Computerized ...