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，ATP依赖的核小体重构的测定来测量ATP依赖的核小体重构活动，免疫纯化INO80或INO80 subcomplexes孵育与ATP和mononucleosomal衬底，它包含一个单核小体定位于216碱基对，32 P标记的DNA片段的一端。将反应产物再进行电泳在天然聚丙烯酰胺凝胶。 <ol><li>以产生32 P标记的，"601"的DNA片段，从放大的pGEM-3Z-601 4包含端定位的601核小体定位序列的216 bp的DNA片段，使用寡核苷酸5&#39;-ACAGGATGTATATATCTGACACGTGCCTGG和5&#39;-AATACTCAAGCTTGGATGCCTGCAG如正向和反向引物。 <ol><li>设置了100微升PCR反应，如表1所述。 </li><li>在96℃，followe 1分钟:使用以下程序进行PCR反应在热循环仪ð45秒，94℃，30秒，57℃，60秒，72℃，30个循环; 7分钟72℃的结局。 </li><li>后的PCR反应结束后，通过无核酸酶的离心柱两次通过该反应产物中除去未掺入的核苷酸。 </li><li>运行在1.2％琼脂糖凝胶上对5μl纯化的PCR产物，以确认PCR反应生成所要的〜216碱基对的DNA片段。 </li><li>稀释5微升纯化的PCR产物20倍，并使用UV分光光度计测量DNA浓度。平均产量〜40毫微克/微升。 </li><li>测量的1微升纯化的PCR产物在闪烁计数器中的放射活性。通过计算CPM / NG估计标记效率。一个成功的标记反应，预计产量601的DNA片段〜15,000 CPM / NG。 </li></ol></li><li>转让Hela细胞的核小体上的标记601的DNA采用连续稀释法5。 <ol><li>混合2皮摩尔的32 P-标记的601的DNA片段6微克的Hela核小体在50μl含1.0 M氯化钠，的10mM Tris-盐酸，pH值8.0，1mM EDTA中，0.1mM的PMSF和1mM的缓冲液（其制备如所述5） DTT和孵育在30℃下进行30分钟。 </li><li>依次稀释该混合物至0.8摩尔，0.6 M和0.4 M氯化钠的10毫摩尔Tris-盐酸（pH值8.0），1mM EDTA中，0.1mM的PMSF由稀释用12.5微升，20.8微升，41.6微升，分别和1mM DTT的，用30分钟温育在30℃下各稀释后。 </li><li>进一步稀释该混合物至0.2M，然后以0.1 M氯化钠加入125微升，然后加入250μl含有0.1％诺乃洗涤剂P-40，20％甘油，和200微克/毫升BSA的相同缓冲液中，用30分钟温育在30℃下的最后两个稀释度之间。储存在4℃的mononucleosome基板长达3个月。 </li></ol></li><li>在10μl反应体系进行ATP依赖的核小体滑动反应。在REAction组分列在表2中，由于最佳NaCl浓度为INO80核小体重构活性为〜50 mM氯化钠（未发表结果），调整NaCl在各反应至50mM的总浓度，考虑到的NaCl中含有的量核小体和酶制剂。 <ol><li>在开始建立测定法，浇铸天然聚丙烯酰胺凝胶（18×16厘米）。以制备含5％丙烯酰胺的单一凝胶（丙烯酰胺:双丙烯酰胺37.5:1），0.5×TBE（45毫摩尔Tris硼酸盐，1mM EDTA）中，0.01％的过硫酸铵（APS）和0.001％的N，N，N&#39;， N&#39;四甲基乙二胺（TEMED），混合在表3中所列的成分。允许凝胶聚合至少2小时，在室温。 </li><li>另一方面，对于将要执行的每个反应中，结合在预冷的硅化1.5 ml离心管〜20nM的INO80或INO80亚复合（其制备如所述3）的EB100缓冲量（ <str翁&gt;表4）足以得到4.75微升的体积。立即重新冻结在粉状干冰或液氮中任何剩余的含INO80级分。 </li><li>设置主鸡尾酒的配料的其余部分，由"X"的因素扩大（其中3 X =总数的反应）。需要一个单一的10微升反应的各成分的量被列于表5中 ;配方应根据待进行的反应的数目按比例增加。通过敲击管道或吹打上下拌匀，用的Pipetman和旋管几秒钟的台式离心。 </li><li>免除5.25微升主鸡尾酒的每个反应管设置在步骤1.3.2。吹打上下拌匀。通过将反应管于30℃的加热块或水浴启动反应，并孵育2小时。 </li><li>同时，准备"移除组合"包含竞争对手的DNA鸡尾酒与核小体，扩大利用X（反应+ 4 x =总数）的一个因素。根据需要，制备1.5微升除去混在单一反应各成分的量被列于表6;配方应相应加大取决于要进行的测定的次数。 </li><li>加入1.5微升去除混合终止反应。拌匀，降速和孵化，在30℃下再30分钟。 </li><li>同时，在4℃下以100伏运行前在垂直电泳单元的非变性聚丙烯酰胺凝胶30分钟，用0.​​5×TBE为电泳缓冲液与下部腔室内部的磁力搅拌棒，以维持恒定的缓冲循环。 </li><li>加载样品，加入2.5微升含3×TBE，30％甘油，0.25％溴酚蓝，和0.25％二甲苯蓝FF加载染料。拌匀后短暂离心样品，并加载到使用装载提示凝胶。 </li><li>运行在200伏的凝胶为4.5小时，在4℃下用缓冲液循环。</li><li>为了检测信号，将凝胶转移到堆栈两片滤纸。包在滤纸上用保鲜膜上方的凝胶，然后将其暴露到存储荧光屏，在4℃下进行所需的时间。 </li><li>扫描用同位素成像扫描仪系统的屏幕，并使用合适的软件进行数据分析。 </li></ol></li></ol> 2 Mononucleosome结合测定为了分析给定INO80复杂，mononucleosomes的结合亲和力，执行使用步骤1.2生成的mononucleosomal基板的电泳迁移率变动分析（EMSA）。 <ol><li>建立了反应的混合所描述的核小体重塑的实验，但省略了反应的ATP和拆卸组合结合分析;孵育在30℃下进行30分钟。 </li><li>加入2.5微升上样染料的各反应混合物中，并适用于含有3.5％的丙烯酰胺（丙烯酰胺非变性聚丙烯酰胺凝胶:双37.5:1），1％甘油，0.5×TBE，0.01％APS，和0.001％TEMED。 </li><li>用0.5×TBE为电泳缓冲液，运行在200伏的凝胶2.5小时，在4℃用缓冲液循环和暴露到存储荧光屏。 </li></ol> 3，DNA和核小体依赖性ATP酶测定在含有20mM的Tris-HCl（pH为7.5），60 mM氯化钠，6.6毫米MgCl 2，0.8毫摩尔EDTA，0.015％诺乃洗涤剂P-40，2.5％甘油，0.1毫克/毫升BSA，5微升反应混合物中进行ATP酶测定法毫摩尔DTT，0.1mM的PMSF，2毫摩尔ATP，产品[α-32 P] ATP的2微居里（3000次/毫摩尔）。对于INO80复杂每个INO80复杂或金额进行检测设置三个平行反应，一个包含EB100缓冲区来衡量DNA-或核无关的ATP酶，一种含闭合环状质粒DNA（5,000个基点，约30纳米）来衡量DNA-依赖性ATP酶，和一种含有宫颈癌寡聚核小体（〜185 nm）的测定核小体依赖性ATP酶。成立于冰上的所有反应。 <oL&gt; <li>对于每个反应，结合10-50纳米的免疫纯化INO80或INO80 subcomplexes用的EB100缓冲器足以得到2.2微升的体积中预先冷却的润滑的1.5 ml离心管中的量。立即重新冻结在粉状干冰或液氮中的任何含INO80级分。 </li><li>设置主鸡尾酒。需要一个单一的反应的各成分的量被列于表7中 。向上扩展的配方通过用因子为3（X + 2）1，其中X =待测定INO80制剂的数量按比例放大。 </li><li>以制备"子鸡尾酒"包含缓冲器只，DNA或核小体，可免除2.5（X + 2）微升主鸡尾酒的成三个独立的管中。添加0.3（X + 2）微升无论是EB100的，闭合环状质粒DNA（1.5微克/微升），或宫颈癌寡（1.5微克/微升），搅拌均匀。 </li><li>分配2.8微升适当子鸡尾酒到含酶的反应管设置在日EP 3.1。吸管轻轻上下混用;避免产生气泡。 </li><li>以启动反应，将反应管转移到30℃的热块。 </li><li>后5，15，30，及温育60分钟后，发现0.5微升每个反应混合物到纤维素聚乙烯薄层色谱（TLC）板（20×10厘米）在一条直线上至少为1.5厘米的距离从底部边缘。立即返回反应管于30℃加热块，使多个时间点可以从一个单一的管。点样后，用干一击机的TLC板。 </li><li>传送的TLC板，以含有足够的0.375磷酸钾（pH值3.5），以使底0.5厘米TLC板上，以在溶液中被淹没的玻璃室中。 </li><li>覆盖所述腔室，并发展到液相的前沿到达TLC板的顶部。立即擦干板完全采用吹塑机。 </li><li>揭露干燥薄层板，在室温下存储荧光屏。扫描用同位素成像扫描仪系统的屏幕，并确定放射性ATP底物和ADP产物的量。 </li><li>计算出的ATP水解的量，用以下公式乘以％的ATP通过ATP的存在于起始反应混合物中的量水解:皮摩尔ATP水解= 10pmol的ATP在起始反应×[ADP /（ATP + ADP）] </li></ol>

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The authors declare that they have no competing financial interests.

以确保核小体重构和ATP酶活性，我们观察到在分析依赖INO80复合物的催化活性，并且不污染重塑和/或ATP酶，我们经常测定核小体重构和INO80复合物的催化活性的版本的ATP酶活性，纯化使用野生型INO80同样地进行平行。阴性对照反应缺少ATP也应测定核小体重构的活性时，以测试污染ATP和/或ATP的独立重塑活动的存在，这可能会复杂化的比较INO80复合物或subcomplexes不同制剂的活性实验解释执行。催化INO80?...

SNF2家庭染色质重塑复合物包括中央SNF2般的ATP酶亚基1,2。一些SNF2状ATP酶的功能单亚基酶，而其他用作较大的多亚基复合物的催化亚基。阐明的分子机制，其中每一个染色质重塑复合物有助于其活动亚基的要求来执行解剖重建过程的生化分析的能力。 ATP依赖的核小体重构的人类INO80复合物和其他染色质重塑酶，可以设想为，与重塑酶，以核小体结合启动一个多步骤的过程，随后激活其DNA和/或核小体依赖性ATP酶，易位于核小体的DNA，和核小体1,2的最终位置改变重塑酶。理解ATP依赖的染色质重塑过程r的分子细节equires重塑反应的染色质重塑复合物在反应中的每个步骤的各个子单元的贡献成单个步骤和定义的清扫。这样的分析需要使用定义的底物分子在体外进行分析核小体重塑和其他活动的能力。 在以前的JOVE协议中，我们描述的程序来生成INO80染色质重塑复合物及其亚与定义亚基组成3。在这里，我们提出了3生化检测，使核小体结合，DNA和核小体的激活ATP酶，而这种复合物相关的核小体重塑活动的定量分析。

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			<td height="21" style="height:21px;">40% Acrylamide/Bis 37.5:1</td>
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			<td height="21" style="height:21px;">Sonicated salmon sperm DNAs&#160;</td>
			<td>GE Healthcare</td>
			<td style="width:147px;">27-4565-01</td>
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			<td height="21" style="height:21px;">10% ammonium persulfate (APS)</td>
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			<td height="24" style="height:24px;">[&#945;-32P] ATP (3000 Ci/mmol)</td>
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			<td style="width:147px;">BLU003H250UC</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:359px;">dCTP, [&#945;-32P]- 6000Ci/mmol</td>
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			<td style="width:147px;">BLU013Z250UC</td>
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			<td height="21" style="height:21px;">PCR thermal cycler PTC 200</td>
			<td>MJ Research</td>
			<td style="width:147px;">PTC 200</td>
			<td style="width:123px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hoefer vertical electrophoresis unit</td>
			<td style="width:212px;">Hoefer</td>
			<td style="width:147px;">SE600X-15-1.5</td>
			<td style="width:123px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">lubricated 1.5ml microcentrifuge tubes&#160;</td>
			<td style="width:212px;">Costar</td>
			<td style="width:147px;">3207</td>
			<td style="width:123px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Storage Phosphor Screen&#160;</td>
			<td>Molecular Dynamics</td>
			<td style="width:147px;">63-0034-79</td>
			<td style="width:123px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">3MM filter paper</td>
			<td>Whatman&#160;</td>
			<td style="width:147px;">28458-005</td>
			<td style="width:123px;">VWR</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Typhoon PhosphorImager&#160;</td>
			<td>GE Healthcare</td>
			<td style="width:147px;">8600</td>
			<td style="width:123px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;">ImageQuant software</td>
			<td>GE Healthcare</td>
			<td style="width:147px;">ver2003.02</td>
			<td style="width:123px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">TLC Glass Plates, PEI-Cellulose F</td>
			<td style="width:212px;">Millipore</td>
			<td style="width:147px;">5725-7</td>
			<td style="width:123px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Immobilon-FL Transfer Membrane 7 x 8.4</td>
			<td style="width:212px;">Millipore</td>
			<td style="width:147px;">IPFL07810</td>
			<td style="width:123px;"></td>
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		<tr height="42">
			<td height="42" style="height:42px;width:359px;">General purpose survey meter with end-window or pancake GM (Geiger-Mueller) probe</td>
			<td style="width:212px;">Biodex</td>
			<td style="width:147px;">Model 14C</td>
			<td style="width:123px;"></td>
		</tr>
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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.

该图显示用于表征INO80活动生化分析，包括核小体的滑动（ 图1）和结合（ 图2）测定法和DNA-或核小体依赖性ATP酶测定（ 图3）的代表性结果。 在图1所示的实验中比较了完整INO80复合物的能力，通过FLAG-IES2或FLAG-INO80E纯化的INO80 subcomplexes或者通过FLAG-Ino80ΔN或Ino80ΔNΔHSA精制，催化mononucleosomes汇集了216 bp的，放射性标记的DNA片段?...

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.

生化分析的分析ATP依赖的染色质重塑酶活性的影响

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

Research

JoVE Journal

Biology

14.7K 次观看.  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.

视频: 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.

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 ...

科研

生物学

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.

斑马鱼脑室注射液

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.

漂白实验（FRAP和FLIP）来衡量生活胚胎干细胞的染色质的蛋白质动力学

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.

两种类型的检测，检测青蛙精子Chemoattraction

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.

配对末端标签测序（嘉PET）测绘染色质相互作用和了解转录调控染色质相互作用分析

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.

在成年斑马鱼的生化分析采血车

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.

提取组织抗原的功能分析

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.

生化和高通量的微观评估的脂肪含量<em&gt;秀丽隐杆线虫</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.

糖原生化滴定<em&gt;在体外</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.

活性检测在培养细胞

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