这项工作是从美国国立精神卫生研究所（5R01MH071476）的赠款支持。

程序： 第一日 1。均质与Douncing缓冲区50-500毫克冷冻验尸报告灰色的事情组织。 ！注意-人体组织必须谨慎处理，严格的安全条件下。它应在BSL - 2或更高的安全标准来处理。 <ol><li>以前解剖，验尸报告的大脑从-80 °，dounce Douncing缓冲区5X大脑体积，并在2.0 ml离心管地方。匹配的样品，并控制对同时处理。 </li></ol> 2。微球菌核酸（MN）消化<ol><li>添加微球菌核酸5U/mL移液器放置在冰之前的向上和向下混合样品，并在解决方案。 *关键的一步-重要的是迅速地做到这一步，因为MN具有行动能力，甚至4 ° C 。 </li><li>孵育7分钟样品在37 ° C。 </li><li> 7分钟的潜伏期后，加入0.5M EDTA浓度为10mm，停止MNase活动。 </li></ol> 3。 Hypotonisation <ol><li>将样品成15毫升猎鹰管。添加10X的样本量为0.2mm EDTA，1 / 2000苯甲脒0.2M和0.1M phenylmethanesulphonylfluoride 1 / 1000的样本量（PMSF）的样本量。后两个化合物被用作蛋白酶抑制剂。 * 关键的一步-重要的是要在所有这些步骤的样品置于冰上。 </li><li>孵育1小时的样品，而震荡每隔10分钟。 </li><li>小时的潜伏期长，加上1 / 2000 3M数码地面电视的样本量，但另一种蛋白酶抑制剂。 </li><li>涡样品再次离心10分钟，在3175离心力在4 ° C *可选步骤-与蛋白质G琼脂糖Precleansing。 <ol><li>取上清液，并提出在新的15毫升猎鹰管。 </li><li>添加500μL蛋白G琼脂糖。 </li><li>在室温为30分钟旋转。 </li><li>在10分钟4000转离心4 ° C。 </li></ol></li><li>分发上清液500μL，使输入控制（只含有基因组DNA），和其余的是分裂成两个含1600μL的样品每管 - 染色质免疫沉淀（ChIP）样品。 </li><li>输入控制是摆在-80 ° CO / N，直到进一步使用。 </li><li>对芯片样品 - 1:10体积10​​XFSB和4μg的抗体，然后涡样品添加和旋转4 ° C过夜。 ！小心-金额和抗体稀释可能需要优化。 </li></ol> 第二日 ！注意！ -开始第二日通过洗涤将用于隔离核小体DNA的蛋白G琼脂糖。由于琼脂糖珠是非常敏感的，它是要切断时移液含有琼脂糖珠的任何解决方案的提示元首。 1。探测DNA的蛋白G琼脂糖珠<ol><li>添加1.6 mL的1XFSB 245μL的G蛋白琼脂糖在2 ml离心管（足够4管）（循环）。 </li><li>分成两个2 mL管和填充每个1X外频高达1.6毫升的解决方案。 </li><li>在室温下旋转30秒和0.1离心力离心30秒。 </li><li>去除上清液，用真空。 </li><li>再次加入1.6毫升1X FSB。旋转的样品为30秒，然后在0.1离心力离心30秒。 </li><li>再次取出上清，并结合1.5毫升1XFSB都管。 </li><li>加入15μL超声鲑鱼精子DNA（10mg/ml的）。 ！注意-这一步，原则上应减少非特异性的免疫沉淀结合珠。然而，这也可能导致误报（人类）的DNA序列。 </li><li>在室温下旋转30分钟，然后0.1离心力离心30秒。 </li><li>去除上清。添加200μL1XFSB。 </li><li>添加到每个芯片样品90μL琼脂糖珠。在4 ° C为1小时旋转。 </li><li>添加到剩余的琼脂糖珠1XFSB 1ML作为阴性对照和旋转4 °为1小时。 </li><li>后一小时的潜伏期长，0.1离心力离心30秒的样品和阴性对照。上清液。 </li></ol> 2。洗衣机的珠子 ！注意！ -洗涤缓冲液必须保持在4℃直至使用，并要使用只有不到一个月以前。 <ol><li>珠每个洗涤液中加入1 mL和旋转，在室温下3分钟。 </li><li> 0.1离心力离心30秒。 </li><li>弃上清采用真空。 </li></ol> *每个洗涤液 - 低盐洗涤液 - 高盐洗涤液</p&gt; -氯化锂溶液- 在室温下1分钟只旋转！ - TE缓冲液（10毫米的Tris，1 mM EDTA的pH值= 8） 3。洗脱 *关键的一步 -每一天的实验，它是被用来新鲜的洗脱液。 <ol><li>每个样品加入250μL新鲜配制洗脱缓冲液。 </li><li>旋转在室温为15分钟，然后离心1分钟，在0.4离心力。 </li><li>保存在2.0毫升的离心管（环）上清。 </li><li>洗脱缓冲液中加入250μL到几秒钟，每个样品由专人旋涡。然后，在mulitvortexer 15分钟的旋涡的样品。 </li><li>在16个区域合作框架RPM离心4分钟，并保存在相同的2.0毫升管（环）上清。 </li></ol> 4。消化蛋白质<ol><li>加入10微升0.5M EDTA，25微升0.8M的Tris - HCl，pH值6.5，10毫克/毫升的蛋白酶K（1 / 200采样），每个芯片的样品。 </li><li>每个输入控制，裂解液中加入蛋白酶K消化（样品缓冲液的1 / 10）和10mg/ml的蛋白酶K（样品缓冲液的1 / 200）。 </li><li>输入和芯片样品在52℃孵育至少3小时。 </li></ol> 5。酚/氯仿抽提 ！引擎盖下的注意-实验要求的酚/氯仿，应当执行。使用丁腈手套时，酚/氯仿。 <ol><li> 3个小时的潜伏期后，我们每个样品中加入500μL酚 - 氯仿。 </li><li>涡所有样品几秒钟，然后离心5分钟的转速在13个区域合作框架。 </li><li>在这一点上，分两个阶段将是现在和我们的顶级阶段的内容感兴趣。取出顶部阶段，并放入一个2 ml离心管。 </li><li>每个样品1.375毫升100％的乙醇添加2μL糖原和50μL3M的醋酸钠的混合物。 </li><li>涡所有样品的蓬勃发展。放置在-80 ° C过夜。 </li></ol> 第三日<ol><li>取出样品的形式-80℃，并置于冰上他们解冻。 </li><li> 10分钟在15离心力离心4 ° C。 </li><li>小心取出上清液，在试管底部，而不会干扰沉淀。 </li><li>每个样品中加入1毫升75％冷乙醇，然后反转的4-6倍。 </li><li> 5分钟在18离心力离心4 ° C。 </li><li>去除上清液，再一次，并允许空气干燥的颗粒。 </li><li>干颗粒溶解于50μL的Tris - HCl，PH8 4MM和储存在-80 ° C，直到进一步的使用。 </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>

这里列出的协议在人类大脑的组蛋白和/或DNA甲基化签名感兴趣的调查是非常有用的的，因为这些染色标记可能不容易死后文物相比，其他类型的修改，包括（组蛋白）的乙酰化和磷酸化1的， 2。死后的大脑，是适合单核小体的准备的研究，在很大程度上仍然连接到核心组蛋白的DNA，至少在与代表自溶的时间间隔（死亡和冻结/储存组织之间的时间）范围内的典型标本，几个小时至1.5天。然而，单?...

解决方案： <ul><li> nChIP Douncing缓冲液：10毫米三，MgCl 2的4毫米，1毫米氯化钙 2调整pH值至7.5 </li><li> 10X外频：50 mM的EDTA，三200毫米，500毫米氯化钠调整pH值至7.5 </li><li>低盐洗涤液：0.1％SDS，1％Triton X - 100的的，2毫米EDTA，20毫米的Tris（pH值8），150毫米氯化钠</li><li>高盐洗涤液：0.1％SDS，1％TRITON X - 100，2 mM的EDTA，20毫米的三（PH 8），500毫米氯化钠</li><li>氯化锂缓冲液：1％的IGEPAL - CA 630，1％去氧胆酸，1毫米EDTA（pH值8），10毫米三（pH值8），氯化锂0.25米</li><li> TE缓冲液（pH值8）：10毫米三，1毫米EDTA </li><li>洗脱缓冲液：0.1M碳酸氢钠，1％SDS </li><li>蛋白酶K消化缓冲液（10X）：1M的Tris，50 mM的EDTA，2％SDS，2M氯化钠调节pH至8.0 </li><li> 3 M醋酸钠：24.61 g无水醋酸钠100毫升灭菌蒸馏水（MW = 82.03）。调整pH值至5.2使用浓醋酸。 </li></ul>

a chromatin assay for human brain tissue

慢性神经精神疾病如精神分裂症，双相情感疾病和自闭症被认为是导致从遗传和环境因素可能会导致基因表达和其他分子病理学遗传学改变相结合。然而，传统上，死后大脑中的表达研究局限于基因或蛋白质的量化。在死后大脑的研究，如在自溶时间的可变性和组织integrities中遇到的限制也可能会影响到任何高阶的染色质结构研究。然而，基因组DNA的核小体的组织，包括的DNA：核心组蛋白具有约束力 - 看来主要是由不同的大脑银行提供的有代表性的样品保存。因此，它是研究在死后大脑中定义的基因位点的核心组蛋白的甲基化模式和其他共价修饰。在这里，我们提出一个简化的本地冻结（从来没有固定的）人类的大脑标本的染色质免疫沉淀（NChIP）协议。微球菌核酸酶的消化脑组织匀浆开始，NChIP由qPCR的三天之内就可以完成。这里介绍的方法应该是有用的，以澄清在正常和病变的人类大脑基因表达的表观遗传机制。

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

A Chromatin Assay for Human Brain Tissue

直到最近，表达对人类大脑的研究仅限于RNA或蛋白质的定量。本文中所描述的染色质免疫沉淀技术，将有可能映射在死后大脑基因表达的组蛋白甲基化和其他后生监管。

一个人脑组织中的染色质含量

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

Research

JoVE Journal

Biology

12.8K 次观看.  University of Massachusetts Medical School. 直到最近，表达对人类大脑的研究仅限于RNA或蛋白质的定量。本文中所描述的染色质免疫沉淀技术，将有可能映射在死后大脑基因表达的组蛋白甲基化和其他后生监管。

视频: 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.

肌动蛋白共沉淀测定;​​ F -肌动蛋白结合蛋白分析

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

科研

生物学

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.

从人类胚胎干细胞的染色质免疫沉淀

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.

从人死后脑组织神经元细胞核分离

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.

在激活人类细胞中的基因表达的动态检测组蛋白修饰染色质免疫沉淀（ChIP）

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.

使用早期的小鼠胚胎组织特异性基因的染色质免疫沉淀检测

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

A System for Culturing Iris Pigment Epithelial Cells to Study Lens Regeneration in Newt

Salamanders like newt and axolotl possess the ability to regenerate many of its lost body parts such as limbs, the tail with spinal cord, eye, brain, heart, the jaw 1. Specifically, newts are unique for its lens regeneration capability. Upon lens removal, IPE cells of the dorsal iris transdifferentiate to lens cells and eventually form a new lens in about a month 2,3. This property of regeneration is never exhibited by the ventral iris cells. The regeneration potential of the iris cells can be studied by making transplants of the in vitro cultured IPE cells. For the culture, the dorsal and ventral iris cells are first isolated from the eye and cultured separately for a time period of 2 weeks (Figure 1). These cultured cells are reaggregated and implanted back to the newt eye. Past studies have shown that the dorsal reaggregate maintains its lens forming capacity whereas the ventral aggregate does not form a lens, recapitulating, thus the in vivo process (Figure 2) 4,5. This system of determining regeneration potential of dorsal and ventral iris cells is very useful in studying the role of genes and proteins involved in lens regeneration.

In newt, the lens regenerates always from the dorsal iris by transdifferentiation of the iris pigmented epithelial cells (IPEs). Here we describe a procedure to culture dorsal and ventral newt IPE cells and their implantation to the newt eye. The implanted cells are then studied by tissue sectioning and immunohistochemistry.

一个系统为培养虹膜色素上皮细胞，研究蝾螈镜头再生

Salamanders like newt and axolotl possess the ability to regenerate many of its lost body parts such as limbs, the tail with spinal cord, eye, brain, ...

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.

染色质免疫沉淀（ChIP）使用果蝇组织

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.

代高通量测序技术（ChIP-seq的高品质的染色质免疫沉淀DNA模板）

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.

产生和人类INO80染色质重塑复合物及其亚纯化

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.

适用于比色金纳米粒子检测方法的选择结构切换适配方法

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