이 작품은 정신 건강의 국립 연구소 (5R01MH071476)에서 부여에 의해 지원되었다.

절차 : 1 일 데이 1. 버퍼 Douncing와 냉동 사후 회색 물질 조직 50-500 MG를 균질. ! 주의 - 인간의 조직은 엄격한 안전 조건 하에서 유의해서 다루어 져야한다. 이것은 BSL - 2 이상의 안전 기준에서 처리되어야합니다. <ol><li> -80에서 이전 해부, 사후 뇌를 꺼내 ° C는 Douncing 버퍼의 배 뇌 용적에서 그들을 다운스, 및 2.0 ML의 microcentrifuge 관에서 개최. 샘플과 일치하고 제어 쌍을 동시에 처리됩니다. </li></ol> 2. Micrococcal Nuclease (MN) 소화 <ol><li> 샘플로 Micrococcal nuclease의 5U/mL를 추가하고 얼음에 넣어 전에 위아래로 pipetting하여 솔루션 내에서 섞는다. * 임계 단계 - MN도 4 행동할 수있는 능력을 가지고 이후 신속하게이 단계를 할 중요 ° C. </li><li> 37 7 분 샘플을 품어 ° C. </li><li> 7 분 부화 후, MNase 활동을 중지 10mM의 농도 0.5M EDTA (에틸렌 다이아 민 테트라 초산)를 추가합니다. </li></ol> 3. Hypotonisation <ol><li> 플레이스 샘플 15 매 ML 튜브로. 0.2mM EDTA (에틸렌 다이아 민 테트라 초산), 1 / 0.2M benzamidine과 0.1M의 phenylmethanesulphonylfluoride 1 / 1000 샘플 볼륨 (PMSF) 2000 샘플 볼륨의 10X 샘플 볼륨을 추가합니다. 후자의 두 화합물은 프로 테아제 억제제로 사용됩니다. * 임계 단계 -이 단계 동안 얼음에 샘플을 유지하는 것이 중요합니다. </li><li> 그것을 10 분마다 vortexing하면서 1 시간 동안 샘플을 품어. </li><li> 시간 긴 인큐베이션의 끝에, 2천분의 1 3M DTT의 샘플 볼륨, 또 다른 프로 테아제 억제제를 추가합니다. </li><li> 와동 샘플은 한 번 더 4 ° C. 10 분 3175 RCF에서 원심 분리기 * 선택 단계 - 단백질 G 아가로 오스와 Precleansing. <ol><li> 뜨는 가지고 새로운 15 ML 팔콘 튜브에 넣어. </li><li> 단백질 G 아가로 오스 500 μL를 추가합니다. </li><li> 30 분 상온에서 회전. </li><li> 4 10 분 4,000 rpm으로 원심 분리기 ° C. </li></ol></li><li> 500 μL를 입력 제어 (단 게놈 DNA를 포함)로 사용되도록 뜨는 배포하고, 나머지는 각각의 샘플 1,600 μL 담긴 두개의 튜브로 나뉩니다 - 염색질 immunoprecipitation (칩) 샘플입니다. </li><li> 입력 제어는 -80에서 배치됩니다 ° CO / N 더욱 사용까지. </li><li> 칩 샘플을 위해 - 그 항체의 10XFSB 및 4μg의 1시 10분 볼륨, 소용돌이 샘플을 추가 4 ° C 야간에 그들을 돌린다. ! 주의 - 항체의 금액 및 희석 최적화가 필요할 수 있습니다. </li></ol> 2 차 주 ! 주의! - nucleosomal DNA를 분리하는 데 사용됩니다 단백질 G 아가로 오스를 세척하여 2 차 일 시작합니다. 아가로 오스 비즈 매우 민감한 때문에, 그것은 아가로 오스 비즈가 포함된 솔루션을 pipetting 때마다 팁을의 머리를 잘라하는 것이 필요합니다. 1. DNA에 단백질 G 아가로 오스 비즈를 프로빙 <ol><li> 2 ML의 microcentrifuge 관에 245 μL 단백질 G - 아가로 오스 (충분한 사 튜브에 대한) (루프)로 1.6 ML 1XFSB를 추가합니다. </li><li> 이 두 ML 튜브 및 리필 1X FSB 각 최대 1.6 ML로 들어가서 해결책을 분리. </li><li> 30 초에 대한 RT에서 회전 및 30 초에 대한 0.1 RCF에서 원심 분리기. </li><li> 진공을 사용하여 표면에 뜨는를 제거합니다. </li><li> 다시 한번 1.6 ML 1X FSB를 추가합니다. 30 초에 대한 0.1 RCF에서 원심 분리기 다음 30 초에 대한 샘플을 회전합니다. </li><li> 다시 뜨는을 제거하고, 1.5 ML 1XFSB로 모두 튜브를 결합. </li><li> 15 μL sonicated 연어 정자 DNA (10mg/mL)을 추가합니다. ! 주의 -이 단계는, 원칙적으로 구슬로 immunoprecipitate가 아닌 특정 바인딩을 줄일 수 있습니다. 그러나, 이것은 또한 (인간)의 DNA 시퀀스의 일부에 대한 잘못된 반응을 초래할 수 있습니다. </li><li> 30 분 RT에서 회전 후, 30 초에 대한 0.1 RCF에서 원심 분리기. </li><li> 뜨는을 제거합니다. 1XFSB 200 μL를 추가합니다. </li><li> 각각의 칩 샘플로 아가로 오스 비즈 90 μL를 추가합니다. 1 시간 4 ° C에서 회전합니다. </li><li> 대조군 역할을하고 4 회전 ° C를 1 시간을 위해 남아있는 아가로 오스 비즈로 1XFSB의 1mL를 추가합니다. </li><li> 시간 동안 배양 후, 30 초에 대한 0.1 RCF에서 샘플과 부정 컨트롤을 원심 분리기. 뜨는 폐기하십시오. </li></ol> 2. 비즈 워싱 ! 주의! - 워싱 버퍼는 ° C를 사용하기 전까지, 그리고 오래된 한 달 미만의 경우에만 사용됩니다 4 보관해야합니다. <ol><li> 비즈 각 세척 용액 1 ML을 추가하고 RT 3 분 돌린다. </li><li> 30 초에 대한 0.1 RCF에서 원심 분리기. </li><li> 뜨는 사용하여 진공을 폐기하십시오. </li></ol> * 각 세척 용액 - 낮은 소금 세척 버퍼 - 하이 소금 세척 버퍼 </p&gt; - 염화 리튬 솔루션 - 단 1 분 RT에서 회전! - TE 버퍼 (10 MM 트리스, 1 ㎜ EDTA (에틸렌 다이아 민 테트라 초산) 산도 = 8) 3. 용리 * 임계 단계 - 그것이 사용할 수있는 날짜에 모든 실험에 대한 신선한 용출 버퍼를 확인합니다. <ol><li> 각 샘플에 신선한 용출 버퍼 250 μL를 추가합니다. </li><li> RT 15 분 회전, 다음 1 분 0.4 RCF에서 원심 분리기. </li><li> 2.0 ML의 microcentrifuge 관 (루프)의 표면에 뜨는를 저장합니다. </li><li> 몇 초 동안 손으로 각각의 샘플 및 와동에 용출 버퍼 250 μL를 추가합니다. 그런 다음, mulitvortexer에서 15 분 동안 와동 샘플. </li><li> 사분 16 RCF rpm으로 원심 분리기와 같은 2.0 ML 튜브 (루프)에 뜨는 저장합니다. </li></ol> 4. 다이제스트 단백질 <ol><li> 각각의 칩 샘플 10 μl 0.5M EDTA (에틸렌 다이아 민 테트라 초산), 25 μl 0.8M 트리스 - HCL, pH를 6.5, 10 MG / ML Proteinase K (2백분의 1 샘플)를 추가합니다. </li><li> 각각의 입력 제어, proteinase K의 소화 (샘플 버퍼의 1 / 10) 및 10mg/ml Proteinase K (샘플 버퍼의 1 / 200)에 대한 용해 버퍼를 추가합니다. </li><li> 적어도 3 시간 52 ° C에서 입력 및 칩 샘플을 품어. </li></ol> 5. 클로로포름 / 페놀 추출 ! 주의 - 실험 클로로포름 / 페놀 필요는 후드 아래에 수행되어야한다. 클로로포름 / 페놀 다룰 때 nitrile 장갑을 사용하십시오. <ol><li> 3 시간 부화 후, 각 시료에 500 μl 페놀 - 클로로포름을 추가합니다. </li><li> 몇 초 동안 소용돌이 모든 샘플은 다음 5 분 13 RCF의 rpm으로 원심 분리기. </li><li> 이 시점에서, 두 단계가 존재하고 우리는 최고 단계의 내용에 관심이 있습니다. 상단 위상을 가지고 2 ML의 microcentrifuge 관에 넣어. </li><li> 100 % 에탄올의 1.375 ML과 함께 각각의 샘플에 3M의 아세트산 나트륨의 글리코겐과 50μL의 2μL의 혼합물을 추가합니다. </li><li> 적극적으로 와동 모든 샘플. -80 ° C 하룻밤에 놓으십시오. </li></ol> 제 3 주 <ol><li> 제거 샘플 양식 -80 ° C와 그들이 해동 위해 얼음에 그들을 놓으십시오. </li><li> 4 10 분 15 RCF에서 원심 분리기 ° C. </li><li> 조심스럽게 튜브 하단의 펠렛을 방해하지 않고 뜨는 제거합니다. </li><li> 각 샘플에 감기의 75 % 에탄올 1 ML 추가 후, 그들에게 4-6 번 반전. </li><li> 4 5 분 18 RCF에서 원심 분리기 ° C. </li><li> 한 번 더 뜨는 제거하고 알약 건조 공기 수 있습니다. </li><li> -80 50 4mm짜리 트리스 - HCL, pH8의 μL 및 저장소에 말린 알약을 디졸브 ° C 이상 사용까지. </li></ol>

<ol>
	<li>Huang, H. S., Matevossian, A., Jiang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Akbarian+S.+Chromatin+immunoprecipitation+in+postmortem+brain.">Akbarian S. Chromatin immunoprecipitation in postmortem brain.</a> J Neurosci Methods. 156, 284-292 .</li><li>Huang, H. S., Matevossian, A., Whittle, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prefrontal+dysfunction+in+schizophrenia+involves+mixed-lineage+leukemia+1-regulated+histone+methylation+at+GABAergic+gene+promoters.">Prefrontal dysfunction in schizophrenia involves mixed-lineage leukemia 1-regulated histone methylation at GABAergic gene promoters.</a> J Neurosci. 27, 11254-11262 .</li><li>O'Neill, L. P., Turner, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunoprecipitation+of+native+chromatin:+NChIP.">Immunoprecipitation of native chromatin: NChIP.</a> Methods. 31, 76-82 .</li><li>Spiteri, E., Konopka, G., Coppola, G., 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=Identification+of+the+transcriptional+targets+of+FOXP2,+a+gene+linked+to+speech+and+language,+in+developing+human+brain.">Identification of the transcriptional targets of FOXP2, a gene linked to speech and language, in developing human brain.</a> Am J Hum Genet. 81, 1144-1157 .</li><li>Huang, H. u. a. n. g., Akbarian, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GAD1+mRNA+expression+and+DNA+methylation+in+prefrontal+cortex+of+subjects+with+schizophrenia.">GAD1 mRNA expression and DNA methylation in prefrontal cortex of subjects with schizophrenia.</a> PLoS ONE. 2, .</li><li>Stadler, F., Kolb, G., Rubusch, L., Baker, S. P., Jones, E. G., Akbarian, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histone+methylation+at+gene+promoters+is+associated+with+developmental+regulation+and+region-specific+expression+of+ionotropic+and+metabotropic+glutamate+receptors+in+human+brain.">Histone methylation at gene promoters is associated with developmental regulation and region-specific expression of ionotropic and metabotropic glutamate receptors in human brain.</a> J Neurochem. 94, 324-336 .</li></ol>

프로토콜이 염색질 표시가 (히스톤) 아세틸화 및 인산화 하나를 포함하여 수정의 다른 유형에 비해 사후 유물이 적은 경향이있을 수 있기 때문에, 인간 두뇌의 히스톤 및 / 또는 DNA methylation 서명에 관심이 조사에 특히 유용합니다 여기에 설명된 2. 사후 두뇌는 모노 nucleosomal 준비 연구 의무이며 DNA가 최소한의 범위에서 일반적으로 대표 autolysis 간격 (죽음과 냉동 / 조직의 저장 사이의 시?...

솔루션 : <ul><li> nChIP Douncing 버퍼 : 10 MM 트리스, 4 MM MgCl 2, 1 MM CaCl 2 7.5 산도를 조절 </li><li> 10X FSB : 50 MM EDTA (에틸렌 다이아 민 테트라 초산), 200 MM 트리스, 500 MM NaCl은 7.5 산도를 조절 </li><li> 낮은 소금 세척 버퍼 : 0.1 % SDS, 1 % 트리톤 X - 100, 2 MM EDTA (에틸렌 다이아 민 테트라 초산), 20 MM 트리스 (산도 8), 150 MM NaCl </li><li> 고 염분 세척 버퍼 : 0.1 % SDS, 1 % 트리톤 X - 100, 2 MM EDTA (에틸렌 다이아 민 테트라 초산), 20 MM 트리스 (수소 이온 농도 8), 500 MM NaCl </li><li> 염화 리튬 버퍼 : 1 % IGEPAL - CA 630, 1 % Deoxycholic 산, 1 MM EDTA (에틸렌 다이아 민 테트라 초산) (산도 8), 10 MM 트리스 (산도 8), 0.25 M LiCl </li><li> TE 버퍼 : 10 MM 트리스 (산도 8), 1 MM EDTA (에틸렌 다이아 민 테트라 초산) </li><li> 용출 버퍼 : 0.1M NaHCO 3, 1 % SDS </li><li> Proteinase K의 소화 버퍼 (10X) : 1M 트리스, 50 MM EDTA (에틸렌 다이아 민 테트라 초산), 2% SDS, 2M NaCl은 8.0 산도를 조절 </li><li> 3 M의 아세트산 나트륨 : autoclaved 증류수 100 ML에서 24.61 g 무수 아세트산 나트륨 (MW가 = 82.03). 집중 아세트산을 사용하여 5.2 산도를 조정합니다. </li></ul>

a chromatin assay for human brain tissue

이러한 정신 분열증, 양극성 질환과 자폐증과 같은 만성 질병 neuropsychiatric은 유전자 발현과 다른 분자 병리의 epigenetic 변경의 결과 수도 유전과 환경 요인의 조합에서 결과로 생각되고 있습니다. 전통적으로 그러나, 사후 두뇌의 표현 연구 mRNA 또는 단백질의 부량에 국한되어 있었다. 이러한 autolysis 시간과 조직 integrities에 variabilities으로 사후 뇌 연구에있어서 제한은 또한 높은 주문 염색질 구조의 연구에 영향을 가능성이 있습니다. 그러나, DNA를 포함한 게놈 DNA의 nucleosomal 조직 : 바인딩 핵심 히스톤은 - 대부분 다양한 뇌 은행에서 제공하는 대표적인 표본의 보존에 나타납니다. 따라서, 그것은 methylation 패턴과 사후 두뇌에 정의된 게놈 loci에서 핵심 histones의 다른 공유 결합 수정을 공부하는 것이 가능합니다. 여기, 우리는 냉동 (- 고정하지 않음) 인간의 두뇌 표본에 대한 간단한 기본 염색질 immunoprecipitation (NChIP) 프로토콜을 제시한다. 두뇌 homogenates의 micrococcal nuclease의 소화를 시작으로, NChIP는 qPCR 다음은 3 일 이내에 완료할 수 있습니다. 여기에 제시 방법은 정상과 병적인 인간의 두뇌에서 유전자 발현의 epigenetic 메커니즘을 명료하게하다하는 데 유용합니다.

Watch this Scientific Journal Video about A Chromatin Assay for Human Brain Tissue at JoVE.com

A Chromatin Assay for Human Brain Tissue

최근까지, 인간의 두뇌에 대한 표현 연구 RNA 또는 단백질의 양을 정함으로 제한했다. 이 문서에 설명되어있는 염색질 immunoprecipitation 기술과 함께, 그것은 사후 두뇌에서 유전자 발현의 히스톤 methylation 및 기타 epigenetic 규제를지도로 가능합니다.

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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12.8K 조회수.  University of Massachusetts Medical School. 최근까지, 인간의 두뇌에 대한 표현 연구 RNA 또는 단백질의 양을 정함으로 제한했다. 이 문서에 설명되어있는 염색질 immunoprecipitation 기술과 함께, 그것은 사후 두뇌에서 유전자 발현의 히스톤 methylation 및 기타 epigenetic 규제를지도로 가능합니다.

비디오: A Chromatin Assay for Human Brain Tissue

Actin Co-Sedimentation Assay; for the Analysis of Protein Binding to F-Actin

The actin cytoskeleton within the cell is a network of actin filaments that allows the movement of cells and cellular processes, and that generates tension and helps maintains cellular shape.  Although the actin cytoskeleton is a rigid structure, it is a dynamic structure that is constantly remodeling.  A number of proteins can bind to the actin cytoskeleton.  The binding of a particular protein to F-actin is often desired to support cell biological observations or to further understand dynamic processes due to remodeling of the actin cytoskeleton. The actin co-sedimentation assay is an in vitro assay routinely used to analyze the binding of specific proteins or protein domains with F-actin.  The basic principles of the assay involve an incubation of the protein of interest (full length or domain of) with F-actin, ultracentrifugation step to pellet F-actin and analysis of the protein co-sedimenting with F-actin.  Actin co-sedimentation assays can be designed accordingly to measure actin binding affinities and in competition assays.

Proteins bind to filamentous actin (F-actin) through distinct actin binding modules. In this video we demonstrate the procedure of actin co-sedimentation, which is an in vitro assay routinely used to analyze proteins or specific domains that bind F-actin.

굴지 공동 침강 분석, F - 굴지에 바인딩 단백질의 분석

The actin cytoskeleton within the cell is a network of actin filaments that allows the movement of cells and cellular processes, and that generates ...

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Chromatin Immunoprecipitation from Human Embryonic Stem Cells

The functional and structural complexity of the myriad of cells in metazoan organisms arises from a small number of stem cells. Stem cells are characterized by two fundamental properties: self-renewal and multipotency that allows a stem cell to differentiate into virtually any cell type 1. The progression stem cell to differentiated cell is characterized by loss of multipotency, structural and morphological changes and the hierarchic activity of transcription factors and signaling molecules, whose activities establish and maintain cell-type specific gene expression patterns. At the molecular level, cell differentiation involves dynamic changes of the structure and composition of chromatin and the detection of those dynamic changes can provide valuable insights into the functional features of stem cells and the cell differentiation process 2,3. Chromatin is a highly compacted DNA-protein complex that forms when cells package chromosomal DNA with proteins, mainly histones 4. Stemcellness and cell differentiation has been correlated with the presence of specific arrays of regulatory proteins such as epigenetic factors, histone variants, and transcription factors 2,3,5.
 Chromatin immunoprecipitation (ChIP) provides a valuable method to monitor the presence of RNA, proteins, and protein modifications in chromatin 6,7. The comparison of chromatin from different cell types can elucidate dynamic changes in protein-chromatin associations that occur during cell differentiation.
Chromatin immunoprecipitation involves the purification of in vivo cross-linked chromatin. The isolated chromatin is reduced to smaller fragments by enzymatic digestion or mechanical force. Chromatin fragments are precipitated using specific antibodies to target proteins or protein and DNA modifications. The precipitated DNA or RNA is purified and used as a template for PCR or DNA microarray based assays. Prerequisites for a successful ChIP are high quality antibodies to the desired antigen and the availability of chromatin from control cells that do not express the target molecule. ChIP can correlate the presence of proteins, protein and RNA modifications, and RNA with specific target DNA, and depending on the choice of outread tool, detects the association of target molecules at specific target genes or in the context of an entire genome. The comparison of the distribution of proteins in the chromatin of differentiating cells can elucidate the dynamic changes of chromatin composition that coincide with the progression of cells along a cell lineage.

The differentiation of ESC coincides with cell-type specific changes in the structure and composition of chromatin. The detection of those changes provides valuable insights into the mechanisms that define stemcellness and cell differentiation. Chromatin immunoprecipitation (ChIP) represents a valuable method to dissect the molecular mechanisms underlying stem cell differentiation.

인간 배아 줄기 세포에서 염색질의 Immunoprecipitation

The functional and structural complexity of the myriad of cells in metazoan organisms arises from a small number of stem cells. Stem cells are ...

Neuronal Nuclei Isolation from Human Postmortem Brain Tissue

Neurons in the human brain become postmitotic largely during prenatal development, and thus maintain their nuclei throughout the full lifespan. However, little is known about changes in neuronal chromatin and nuclear organization during the course of development and aging, or in chronic neuropsychiatric disease. However, to date most chromatin and DNA based assays (other than FISH) lack single cell resolution. To this end, the considerable cellular heterogeneity of brain tissue poses a significant limitation, because typically various subpopulations of neurons are intermingled with different types of glia and other non-neuronal cells. One possible solution would be to grow cell-type specific cultures, but most CNS cells, including neurons, are ex vivo sustainable, at best, for only a few weeks and thus would provide an incomplete model for epigenetic mechanisms potentially operating across the full lifespan.  Here, we provide a protocol to extract and purify nuclei from frozen (never fixed) human postmortem brain. The method involves extraction of nuclei in hypotonic lysis buffer, followed by ultracentrifugation and immunotagging with anti-NeuN antibody. Labeled neuronal nuclei are then collected separately using fluorescence-activated sorting. This method should be applicable to any brain region in a wide range of species and suitable for chromatin immunoprecipitation studies with site- and modification-specific anti-histone antibodies, and for DNA methylation and other assays. 

The cellular heterogeneity of brain tissue poses a significant limitation for the study of epigenetic markings in chromatin because most assays lack single cell resolution. Neurons typically are intermingled with glia and other non-neuronal cells. We provide a protocol to extract and collect neuronal nuclei from human brain.

인간의 사후 뇌 조직에서 핵의 연결을 격리

Neurons in the human brain become postmitotic largely during prenatal development, and thus maintain their nuclei throughout the full lifespan. However, ...

Chromatin Immunoprecipitation (ChIP) to Assay Dynamic Histone Modification in Activated Gene Expression in Human Cells

In response to a variety of extracellular ligands, the STAT (signal transducer and activator of transcription) transcription factors are rapidly recruited from their latent state in the cytoplasm to cell surface receptors where they are activated by phosphorylation at a single tyrosine residue1. They then dimerize and translocate to the nucleus to drive the transcription of target genes, affecting growth, differentiation, homeostasis and the immune response. Not surprisingly, given their widespread involvement in normal cell processes, dysregulation of STAT function contributes to human disease, particularly to cancers2 and autoimmune diseases3. It is well established that transcription is regulated by alterations to the chromatin template4,5. These alterations include the activities of ATP-dependent complexes, as well as covalent histone modifications and DNA methylation6. Because STAT activation of gene expression is both rapid and transient, it requires specific mechanisms for modulating the chromatin template at STAT-dependent gene loci. To define these mechanisms, we characterize the histone modifications and the enzymatic activities that generate them at gene loci that respond to STAT signaling. This protocol describes chromatin immunoprecipitation, a method that is valuable for the study of STAT signaling to chromatin in activated gene expression.

Chromatin Immunoprecipitation ChIP to Assay Dynamic Histone Modification in Activated Gene Expression in Human Cells

This protocol describes how chromatin immunoprecipitation (ChIP) is used to study the dynamic alterations to the chromatin template that regulate transcription induced by a signal transduction pathway.

인간의 세포에서 활성 유전자 발현의 어세 동적 히스톤 수정을 염색질 Immunoprecipitation (칩)

In response to a variety of extracellular ligands, the STAT (signal transducer and activator of transcription) transcription factors are rapidly recruited ...

Chromatin Immunoprecipitation Assay for Tissue-specific Genes using Early-stage Mouse Embryos

Chromatin immunoprecipitation (ChIP) is a powerful tool to identify protein:chromatin interactions that occur in the context of living cells 1-3. This technique has been widely exploited in tissue culture cells, and to a lesser extent, in primary tissue. The application of ChIP to rodent embryonic tissue, especially at early times of development, is complicated by the limited amount of tissue and the heterogeneity of cell and tissue types in the embryo. Here we present a method to perform ChIP using a dissociated embryonic day 8.5 (E8.5) embryo. Sheared chromatin from a single E8.5 embryo can be divided into up to five aliquots, which allows the investigator sufficient material for controls and for investigation of specific protein:chromatin interactions.
We have utilized this technique to begin to document protein:chromatin interactions during the specification of tissue-specific gene expression programs. The heterogeneity of cell types in an embryo necessarily restricts the application of this technique because the result is the detection of protein:chromatin interactions without distinguishing whether the interactions occur in all, a subset of, or a single cell type(s). However, examination of tissue-specific genes during or following the onset of tissue-specific gene expression is feasible for two reasons. First, immunoprecipitation of tissue specific factors necessarily isolates chromatin from the cell type where the factor is expressed. Second, immunoprecipitation of coactivators and histones containing post-translational modifications that are associated with gene activation should only be found at genes and gene regulatory sequences in the cell type where the gene is being or has been activated. The technique should be applicable to the study of most tissue-specific gene activation events.
In the example described below, we utilized E8.5 and E9.5 mouse embryos to examine factor binding at a skeletal muscle specific gene promoter. Somites, which are the precursor tissues from which the skeletal muscles of the trunk and limbs will form, are present at E8.5-9.54,5. Myogenin is a regulatory factor required for skeletal muscle differentiation 6-9. The data demonstrate that myogenin is associated with its own promoter in E8.5 and E9.5 embryos. Because myogenin is only expressed in somites at this stage of development 6,10, the data indicate that myogenin interactions with its own promoter have already occurred in skeletal muscle precursor cells in E8.5 embryos.

We demonstrate a chromatin immunoprecipitation (ChIP) method to identify factor interactions at tissue-specific genes during or after the onset of tissue-specific gene expression in mouse embryonic tissue. This protocol should be widely applicable for the study of tissue-specific gene activation as it occurs during normal embryonic development.

초기 마우스 배아를 사용하는 조직 특정 유전자에 대한 염색질의 Immunoprecipitation 분석

Chromatin immunoprecipitation (ChIP) is a powerful tool to identify protein:chromatin interactions that occur in the context of living cells 1-3. This ...

Chromatin Immunoprecipitation (ChIP) using Drosophila tissue

Epigenetics remains a rapidly developing field that studies how the chromatin state contributes to differential gene expression in distinct cell types at different developmental stages. Epigenetic regulation contributes to a broad spectrum of biological processes, including cellular differentiation during embryonic development and homeostasis in adulthood. A critical strategy in epigenetic studies is to examine how various histone modifications and chromatin factors regulate gene expression. To address this, Chromatin Immunoprecipitation (ChIP) is used widely to obtain a snapshot of the association of particular factors with DNA in the cells of interest.
ChIP technique commonly uses cultured cells as starting material, which can be obtained in abundance and homogeneity to generate reproducible data. However, there are several caveats: First, the environment to grow cells in Petri dish is different from that in vivo, thus may not reflect the endogenous chromatin state of cells in a living organism. Second, not all types of cells can be cultured ex vivo. There are only a limited number of cell lines, from which people can obtain enough material for ChIP assay.
Here we describe a method to do ChIP experiment using Drosophila tissues. The starting material is dissected tissue from a living animal, thus can accurately reflect the endogenous chromatin state. The adaptability of this method with many different types of tissue will allow researchers to address a lot more biologically relevant questions regarding epigenetic regulation in vivo1, 2. Combining this method with high-throughput sequencing (ChIP-seq) will further allow researchers to obtain an epigenomic landscape.

Chromatin Immunoprecipitation ChIP using Drosophila tissue

Recently high-throughput sequencing technology has greatly increased sensitivity of Chromatin Immunoprecipitation (ChIP) experiment and prompted its application using purified cells or dissected tissue. Here we delineate a method to use ChIP technique with Drosophila tissue, which can address the endogenous chromatin state in a well-characterized biological system.

사용 염색질 Immunoprecipitation (CHIP) Drosophila 조직

Epigenetics remains a rapidly developing field that studies how the chromatin state contributes to differential gene expression in distinct cell types at ...

Generation of High Quality Chromatin Immunoprecipitation DNA Template for High-throughput Sequencing (ChIP-seq)

ChIP-sequencing (ChIP-seq) methods directly offer whole-genome coverage, where combining chromatin immunoprecipitation (ChIP) and massively parallel sequencing can be utilized to identify the repertoire of mammalian DNA sequences bound by transcription factors in vivo. "Next-generation" genome sequencing technologies provide 1-2 orders of magnitude increase in the amount of sequence that can be cost-effectively generated over older technologies thus allowing for ChIP-seq methods to directly provide whole-genome coverage for effective profiling of mammalian protein-DNA interactions.
For successful ChIP-seq approaches, one must generate high quality ChIP DNA template to obtain the best sequencing outcomes. The description is based around experience with the protein product of the gene most strongly implicated in the pathogenesis of type 2 diabetes, namely the transcription factor transcription factor 7-like 2 (TCF7L2). This factor has also been implicated in various cancers.
Outlined is how to generate high quality ChIP DNA template derived from the colorectal carcinoma cell line, HCT116, in order to build a high-resolution map through sequencing to determine the genes bound by TCF7L2, giving further insight in to its key role in the pathogenesis of complex traits.

Generation of High Quality Chromatin Immunoprecipitation DNA Template for High-throughput Sequencing ChIP-seq

The combination of chromatin immunoprecipitation and ultra-high-throughput sequencing (ChIP-seq) can identify and map protein-DNA interactions in a given tissue or cell line. Outlined is how to generate a high quality ChIP template for subsequent sequencing, using experience with the transcription factor TCF7L2 as an example.

높은 처리량 시퀀싱을위한 고품질 염색질 면역 침전의 DNA 템플릿의 생성 (칩 SEQ)

ChIP-sequencing (ChIP-seq) methods directly  offer whole-genome coverage, where combining chromatin immunoprecipitation  (ChIP) and massively parallel ...

Generation and Purification of Human INO80 Chromatin Remodeling Complexes and Subcomplexes

INO80 chromatin remodeling complexes regulate nucleosome dynamics and DNA accessibility by catalyzing ATP-dependent nucleosome remodeling. Human INO80 complexes consist of 14 protein subunits including Ino80, a SNF2-like ATPase, which serves both as the catalytic subunit and the scaffold for assembly of the complexes. Functions of the other subunits and the mechanisms by which they contribute to the INO80 complex&#39;s chromatin remodeling activity remain poorly understood, in part due to the challenge of generating INO80 subassemblies in human cells or heterologous expression systems. This JOVE protocol describes a procedure that allows purification of human INO80 chromatin remodeling subcomplexes that are lacking a subunit or a subset of subunits. N-terminally FLAG epitope tagged Ino80 cDNA are stably introduced into human embryonic kidney (HEK) 293 cell lines using Flp-mediated recombination. In the event that a subset of subunits of the INO80 complex is to be deleted, one expresses instead mutant Ino80 proteins that lack the platform needed for assembly of those subunits. In the event an individual subunit is to be depleted, one transfects siRNAs targeting this subunit into an HEK 293 cell line stably expressing FLAG tagged Ino80 ATPase. Nuclear extracts are prepared, and FLAG immunoprecipitation is performed to enrich protein fractions containing Ino80 derivatives. The compositions of purified INO80 subcomplexes can then be analyzed using methods such as immunoblotting, silver staining, and mass spectrometry. The INO80 and INO80 subcomplexes generated according to this protocol can be further analyzed using various biochemical assays, which are described in the accompanying JOVE protocol. The methods described here can be adapted for studies of the structural and functional properties of any mammalian multi-subunit chromatin remodeling and modifying complexes.

This protocol describes a procedure for generating and purifying wild type and mutant versions of the human INO80 chromatin remodeling complex. Epitope tagged versions of INO80 subunits are stably expressed in HEK293 cells, and complete complexes and complexes lacking specific sets of subunits are purified by immunoaffinity chromatography.

세대와 인간의 INO80 염색질 리모델링 단지 및 서브 콤플렉스의 정화

INO80 chromatin remodeling complexes regulate nucleosome dynamics and DNA accessibility by catalyzing ATP-dependent nucleosome remodeling. Human INO80 ...

A Method for Selecting Structure-switching Aptamers Applied to a Colorimetric Gold Nanoparticle Assay

Small molecules provide rich targets for biosensing applications due to their physiological implications as biomarkers of various aspects of human health and performance. Nucleic acid aptamers have been increasingly applied as recognition elements on biosensor platforms, but selecting aptamers toward small molecule targets requires special design considerations. This work describes modification and critical steps of a method designed to select structure-switching aptamers to small molecule targets. Binding sequences from a DNA library hybridized to complementary DNA capture probes on magnetic beads are separated from nonbinders via a target-induced change in conformation. This method is advantageous because sequences binding the support matrix (beads) will not be further amplified, and it does not require immobilization of the target molecule. However, the melting temperature of the capture probe and library is kept at or slightly above RT, such that sequences that dehybridize based on thermodynamics will also be present in the supernatant solution. This effectively limits the partitioning efficiency (ability to separate target binding sequences from nonbinders), and therefore many selection rounds will be required to remove background sequences. The reported method differs from previous structure-switching aptamer selections due to implementation of negative selection steps, simplified enrichment monitoring, and extension of the length of the capture probe following selection enrichment to provide enhanced stringency. The selected structure-switching aptamers are advantageous in a gold nanoparticle assay platform that reports the presence of a target molecule by the conformational change of the aptamer. The gold nanoparticle assay was applied because it provides a simple, rapid colorimetric readout that is beneficial in a clinical or deployed environment. Design and optimization considerations are presented for the assay as proof-of-principle work in buffer to provide a foundation for further extension of the work toward small molecule biosensing in physiological fluids.

A protocol is provided to select structure-switching aptamers for small molecule targets based on a tunable stringency magnetic bead selection method. Aptamers selected with structure-switching properties are beneficial for assays that require a conformational change to signal the presence of a target, such as the described gold nanoparticle assay.

색채 금 나노 입자 분석에 적용 구조 전환 앱 타머를 선택하는 방법

Small molecules provide rich targets for biosensing applications due to their physiological implications as biomarkers of various aspects of human health ...

A Cell Free Assay to Study Chromatin Decondensation at the End of Mitosis

During the vertebrate cell cycle chromatin undergoes extensive structural and functional changes. Upon mitotic entry, it massively condenses into rod shaped chromosomes which are moved individually by the mitotic spindle apparatus. Mitotic chromatin condensation yields chromosomes compacted fifty-fold denser as in interphase. During exit from mitosis, chromosomes have to re-establish their functional interphase state, which is enclosed by a nuclear envelope and is competent for replication and transcription. The decondensation process is morphologically well described, but in molecular terms poorly understood: We lack knowledge about the underlying molecular events and to a large extent the factors involved as well as their regulation. We describe here a cell-free system that faithfully recapitulates chromatin decondensation in vitro, based on mitotic chromatin clusters purified from synchronized HeLa cells and X. laevis egg extract. Our cell-free system provides an important tool for further molecular characterization of chromatin decondensation and its co-ordination with processes simultaneously occurring during mitotic exit such as nuclear envelope and pore complex re-assembly.

The molecular mechanisms of the decondensation of highly compacted mitotic chromatin are ill-defined. We present a cell-free assay based on mitotic chromatin clusters isolated from HeLa cells and Xenopus laevis egg extract that faithfully reconstitutes the decondensation process in vitro.

셀 무료 분석은 유사 분열의 끝에서 염색질 Decondensation을 공부하기

During the vertebrate cell cycle chromatin undergoes extensive structural and functional changes. Upon mitotic entry, it massively condenses into rod ...