我们感谢蒂埃里Pourcher医生允许我们使用荧光光谱仪和他的帮助。该实验室是由法甲驳国立癌症勒（队报labellisée）资助，该协会倒拉RECHERCHE CONTRE乐巨蟹研究所国家杜癌症（INCA），在国立新电倒拉RECHERCHE，METOXIA（欧盟第七框架计划），该中心A.拉卡萨涅，该中心法国国家科学研究，研究所国家德拉桑特等德拉RECHERCHEMédicale和尼斯大学（ <a href="http://www.unice.fr/isdbc/" target="_blank">http://www.unice.fr/isdbc/</a> ）。我们感谢博士M克里斯蒂安卜拉希米 - 霍恩的批判性阅读和编辑更正。

糖原生化滴定 1。细胞裂解<ol><li>种子细胞以0.5-2×10 6个，每100毫米直径的培养皿的浓度。 </li><li>治疗方法:细胞培养24，48，或72小时的氧气浓度低（缺氧）的一个Bug盒厌氧工作站（Ruskinn科技Biotrace INTERNATIONAL PLC，英国Bridgend）定为1％或0.1％的O 2，94％或94.9％N 2和5％CO 2。同时，培养细胞在常氧（21％O2，5％CO2）。改变培养基（25mM的含有葡萄糖的培养基中），每24小时以在实验的时间变化最小的葡萄糖浓度。 </li><li>治疗后，用PBS洗涤细胞2倍，从培养基中除去葡萄糖的痕迹。刮去细胞冷PBS。 </li><li>离心溶液以1,000 rpm离心5分钟，弃去上清，用PBS洗涤沉淀一次。沉淀可在-20°C进一步处理之前被冻结。</li><li>重悬沉淀在100μl蒸馏水中。加入100μl的糖原水解缓冲器提供与糖原测定试剂盒（Abcam公司）。煮沸裂解物在95℃下5分钟，以灭活酶。 </li><li>涡旋并离心溶胞产物以13,000 rpm离心10分钟以除去不溶性产物。保留上清液。 </li><li>正常化糖原水平到样本之间的蛋白质含量，蛋白质的定量可以用25-35微升使用二喹啉甲酸测定（BCA-INTERCHIM）的上清液进行。 </li></ol>一些裂解物可用于测量细胞内的游离葡萄糖浓度。 2。糖原水解<ol><li>混合50微升上清液（步骤1）与1微升的水解酶混合物的。该混合物中含有淀粉酶。孵育30分钟，在室温下进行。 </li><li>通过稀释标准糖原制备校准曲线（2毫克/毫升）■upplied与试剂盒，以1​​0微克/毫升在水解缓冲液的溶液。接着，准备6 0分离的管，4，8，12，16，和20微升的10微克/毫升的标准和每个管调整到50微升与水解缓冲液的最终体积，得到的0糖原的浓度，0.04 ，0.08，0.12，0.16，和0.2微克/毫升。加入1μl水解酶混合物的标准中并孵育30分钟，在室温下进行。 </li></ol>该细胞系的背景葡萄糖浓度，按值的游离葡萄糖浓度给出，必须先减去该值的总葡萄糖浓度（从糖原水解+游离葡萄糖葡萄糖）来确定糖原在细胞中的水平。 3。发展与阅读荧光<ol><li>对于每个条件，（管1）准备一管来测量游离葡萄糖浓度和两管（管2和3）来测量总的葡萄糖浓度（每管机智哈哈不同的卷水解糖原）。 </li><li>加15微升上清液在管1步骤1的端部抽出。加35微升的水解缓冲液来完成的，以50微升的最终体积。得到的荧光将得到游离葡萄糖浓度。 </li><li>在管2添加水解糖原（在步骤2中获得的） 第 X微升。调整到50微升的最终体积。样品的体积（倍微升）必须根据为了获得所没有超出校准曲线的浓度的荧光值的细胞密度和/或细胞类型进行调整。 </li><li>添加水解糖原的2×μl，以管3。调整到50微升的最终体积。 </li><li>通过将每个管48.7微升发展缓冲区，1微升开发酶混合物和0.3微升OxiRed探头（在糖原检测试剂盒提供）准备样品和标准开发的解决方案。例如，对于2的条件下，准备一个混合的12管S（6标准，2个免费的葡萄糖和4总葡萄糖荧光）。 </li><li>加50μl的此混合物至每个管并孵育30分钟，在室温下在黑暗中。 </li><li>测量的荧光在535纳米，587纳米的发射波长的激发波长的建议，并为3nm用于激发和发射的狭缝。 </li></ol>

<ol>
	<li>Pelletier, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glycogen+Synthesis+is+Induced+in+Hypoxia+by+the+Hypoxia-Inducible+Factor+and+Promotes+Cancer+Cell+Survival.">Glycogen Synthesis is Induced in Hypoxia by the Hypoxia-Inducible Factor and Promotes Cancer Cell Survival.</a> Front Oncol. 2, (2012).</li><li>Alonso, M. D., Lomako, J., Lomako, W. M., Whelan, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+look+at+the+biogenesis+of+glycogen.">A new look at the biogenesis of glycogen.</a> FASEB J. 9, 1126-1137 (1995).</li><li>Bollen, M., Keppens, S., Stalmans, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specific+features+of+glycogen+metabolism+in+the+liver.">Specific features of glycogen metabolism in the liver.</a> Biochem. J. 336 (Pt 1), 19-31 (1998).</li><li>Parker, G. J., Koay, A., Gilbert-Wilson, R., Waddington, L. J., Stapleton, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AMP-activated+protein+kinase+does+not+associate+with+glycogen+alpha-particles+from+rat+liver.">AMP-activated protein kinase does not associate with glycogen alpha-particles from rat liver.</a> Biochem. Biophys. Res. Commun. 362, 811-815 (2007).</li><li>Sullivan, M. A., 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=Nature+of+alpha+and+beta+particles+in+glycogen+using+molecular+size+distributions.">Nature of alpha and beta particles in glycogen using molecular size distributions.</a> Biomacromolecules. 11, 1094-1100 (2010).</li><li>Chee, N. P., Geddes, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+structure+of+liver+glycogen.">The structure of liver glycogen.</a> FEBS Lett. 73, 164-166 (1977).</li><li>Roach, P. J., Depaoli-Roach, A. A., Hurley, T. D., Tagliabracci, V. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glycogen+and+its+metabolism:+some+new+developments+and+old+themes.">Glycogen and its metabolism: some new developments and old themes.</a> Biochem. J. 441, 763-787 (2012).</li><li>Moses, S. W., Bashan, N., Gutman, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glycogen+metabolism+in+the+normal+red+blood+cell.">Glycogen metabolism in the normal red blood cell.</a> Blood. 40, 836-843 (1972).</li><li>Agbanyo, M., Taylor, N. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Incorporation+of+3-deoxy-3-fluoro-D-glucose+into+glycogen+and+trehalose+in+fat+body+and+flight+muscle+in+Locusta+migratoria.">Incorporation of 3-deoxy-3-fluoro-D-glucose into glycogen and trehalose in fat body and flight muscle in Locusta migratoria.</a> Biosci. Rep. 6, 309-316 (1986).</li><li>Louzao, M. C., 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="Fluorescent+glycogen"+formation+with+sensibility+for+in+vivo+and+in+vitro+detection.">"Fluorescent glycogen" formation with sensibility for in vivo and in vitro detection.</a> Glycoconj. J. 25, 503-510 (2008).</li><li>Sheehan, D. C. H., B, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Theory and practice of histotechnology. , (1980).</li><li>Baba, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+monoclonal+antibody+that+recognizes+glycogen+and+its+application+for+immunohistochemistry.">Production of monoclonal antibody that recognizes glycogen and its application for immunohistochemistry.</a> Kokubyo Gakkai Zasshi. 60, 264-287 (1993).</li></ol>

作者宣称没有利益冲突。

糖原在体外生化滴定允许细胞糖原含量的准确定量。比较一些其他技术（PAS，免疫荧光与糖原抗体等 ），该滴定是非常特异，灵敏，可重复的。此外，该方法是方便的，因为它不需要放射性但荧光光谱仪。然而，这种技术是纯粹的定量和不提供有关在细胞糖原分布的信息。 在这个手稿中描述的技术是实现对细胞提取物中，但它也可以实现对组织切片以用于从组?...

糖原是葡萄糖残基的多分支聚合物，其存在于许多类型的细胞的细胞质中。它的能量储存在细胞中的主要形式之一，在糖代谢中起重要作用。大多数哺乳动物细胞能够生产和储存的糖原，可迅速降解成葡萄糖代谢应激过程中，以促进糖酵解和ATP生产。肝细胞糖原产生大量的调节血糖水平，从而提供给身体一个连续供应的葡萄糖。与此相反，糖原在其它细胞（肌肉，红细胞等 ）的浓度相对较低。然而，在当地，这些量足以在短期内，当细胞被突然暴露于被剥夺营养物质的环境中提供能量。 糖原合成和糖原分解如下在所有组织中相同的步骤（ 图1）。首先，葡萄糖进入细胞的葡萄糖转运蛋白（生产过剩）和葡萄糖-6 - 磷酸（G6P）由磷酸变迅速转化成葡萄糖-1 - 磷酸（G1P）。 G1P然后修改成UDP-葡萄糖，和UDP-葡萄糖的碳C1被附加到糖原生成，糖原的锚定蛋白的酪氨酸残基。这种分子，被认为是糖原引物，由附着了UDP-葡萄糖到终端葡萄糖的延长通过糖原合成酶的α（1→4）键。最后，当形成超过11个葡萄糖残基的直链，分支酶转移一个终端寡糖附近通过α（1→6）​​键由至少6个葡萄糖残基组成的链的另一个葡萄糖。这一过程的重复给出包含形成与每转6.5葡萄糖螺旋分支大规模分形结构。糖原可以反向水解由协调一致的行动，以葡萄糖脱支该水解α（1→6） ​​约束和糖原磷酸化酶能够水解的α（1→4）糖苷结合的一个分支的葡萄糖的最后一个残基与糖原分子之间的酶。此反应称为糖原分解是通过增加AMP水平（反映ATP消耗）激活，并且由葡萄糖和ATP 2,3抑制。 通过电子显微镜，糖原分子已被许多细胞类型中，直径15-30纳米，β自由粒子（或糖原monoparticles）中所述。在特定的细胞类型，例如肝细胞，β粒子可被组装成一个复合物形成玫瑰花结，也称为α粒子，在不同的直径从80纳米至最多为200nm的4 </SUP&gt;。这些β粒子结合形成较大的α粒子的集群的方式还没有完全阐明。一些证据倾向于证明β粒子可以通过共价键5，氢键，或什至通过蛋白质-蛋白质相互作用6键合。糖原储存在细胞的量取决于许多参数:（Ⅰ）在糖原生成发起糖原合成的细胞的数量;由蛋白质磷酸化/去磷酸化调节糖原合酶和磷酸化酶（Ⅱ）的活性;（Ⅲ）的浓度葡萄糖在细胞内，这是依赖于几个参数，如葡萄糖从血管系统的供应和葡萄糖被细胞吸收。肝糖的储存是通过中间代谢产物严格的监管由生物合成的激素变构调节，受激素调节能量代谢，并通过营养感应信号通路7。 是很重要的能够量化的糖原生物样品中更好地了解糖原代谢的整个身体和细胞水平的重要性。我们在这里描述的糖原体外测定准确，重现性好，方便生化。这个技术是基于之前和之后的糖原水解特定的量化葡萄糖荧光。 其他方法存在估计糖原水平在细胞内，但其中大多数是不定量的。一种用于糖原在细胞的定量描述的第一方法是基于[14 C]-葡萄糖掺入糖原8,9的测量值。使用的放射性使得这一过程更难以处理，但它具有提供葡萄糖掺入率成糖原和葡萄糖残基上的外分支和在该分子的核心分布之间进行区分的优点（它也需要一个额外的β-amylolySIS和层析步骤）。另一种技术被开发，最近，是基于2-NBDG的掺入对（2 - {N-[7 - 硝基苯并-2 - 氧杂-1,3 - 二唑-4 - 基]氨基} -2 - 脱氧葡萄糖），一2 -脱氧葡萄糖荧光衍生物，成糖原10。所测量的荧光强度反映了糖原的产生量，并可以用荧光阅读器进行测定。荧光在细胞中的分布，也可以通过共聚焦显微镜评价。 在其他的非定量技术，高碘酸 - 希夫染色（PAS）可能是最常见的。它可用于糖原在固定的细胞，组织切片中的石蜡或冰冻的检测。这种组织学技术的色彩非特异性多糖，糖脂，糖蛋白，纤维素和中性粘蛋白在紫色。本试验的特异性可提高治疗固定细胞或组织切片与淀粉酶，其特异性地消化糖原。此后，糖原的水平可以通过比较未水解样品（所有碳水化合物大分子修饰），以水解样品（碳水化合物大分子修饰糖原除外）进行定性估计。 PAS染色和显微镜分析，与糖原的生化分析，提供关于糖原在细胞中的分布，从而可以扩散或集中在细胞的特定部分的信息。然而，即使在不同条件之间的糖原累积PAS染色估计的差异，这是不定量11。 单克隆抗体的小鼠最初做用下颌骨髁状突软骨作为抗原已被证明与糖原的细胞和用纯化糖原在体外 12反应。作为该抗体特异性地识别糖原相关的糖链，它是糖原在比PAS染色的更具体的方式的检测用免疫组织化学的有用工具。 </p&gt; 电子显微镜是另一种技术，它允许在细胞和糖原贮积的程度的评价晶粒糖原的可视化。事实上，糖原β粒子很容易辨认用电子显微镜作为电子致密颗粒1。

<table border="1" fo:keep-together.within-page="always">
		<tbody>
		 <tr>
			<td></td>
			<td></td>
			<td></td>
			<td>REAGENTS</td>
		 </tr>
		 <tr>
			<td>DMEM</td>
			<td>Invitrogen</td>
			<td>31966.047</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Glycogen Assay Kit</td>
			<td>Abcam</td>
			<td>ab65620 </td>
			<td></td>
		 </tr>
		 <tr>
			<td>PBS</td>
			<td></td>
			<td></td>
			<td></td>
		 </tr>
		<tr>
 	<td></td>
 <td></td>
 <td></td>
 <td>EQUIPMENT</td>
	 </tr>
		 <tr>
			<td>Material Name</td>
			<td>Company</td>
			<td>Catalogue Number</td>
			<td>Comments (optional)</td>
		 </tr>
		 <tr>
			<td>Fluorescence Spectrometer</td>
			<td>PerkinElmer</td>
			<td>LS 50B</td>
			<td></td>
		 </tr>
		</tbody>
 </table>

biochemical titration of glycogen in vitro

糖原是葡萄糖在脊椎动物的主要精力充沛的聚合物和起着全身代谢以及细胞代谢至关重要的作用。许多方法来检测糖原已经存在，但只有少数是定量的。我们在这里描述使用Abcam公司糖原测定试剂盒，它是基于糖原的特异性降解由葡糖淀粉酶对葡萄糖的方法。葡萄糖被再具体地说被氧化，因为与OxiRed探针，以产生荧光反应产物。滴定法准确，灵敏，可在细胞提取物或组织切片来实现。然而，相对于其他技术，它不提供有关糖原在细胞中分布的信息。作为这种技术的一个例子，我们在这里描述的糖原滴定在两种细胞系，中国仓鼠肺成纤维细胞CCL39和人结肠癌LS174，孵育在常氧（21％O 2）与低氧（1％O 2）。我们假设，缺氧是SIGNAL，准备细胞合成和储存糖原为了生存1。

氧气（缺氧）肿瘤中的信号对肿瘤细胞需要存储能量来处理后续的养分耗竭，以便生存的低水平。糖原是葡萄糖在哺乳动物细胞中的主要高能聚合物，我们研究了糖原储存在缺氧的调节。糖原的细胞裂解液中的浓度的计算和标准化，必须对荧光的原始数据，进行如表1，2，和3。的生物化学测定糖原证实，电子致密聚集体上CCL39细胞的电子显微镜照片观察到（ 图2A）</...

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Biochemical Titration of Glycogen In vitro

我们在这里描述的糖原体外滴定准确，重现性好，方便的生化方法。这种技术使用了Abcam公司糖原测定试剂盒和基于糖原的连续水解的荧光成葡萄糖和葡萄糖滴定。

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 ...

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Biochemical Titration of Glycogen In vitro

28.3K 次观看.  University of Nice - Sophia Antipolis. 我们在这里描述的糖原体外滴定准确，重现性好，方便的生化方法。这种技术使用了Abcam公司糖原测定试剂盒和基于糖原的连续水解的荧光成葡萄糖和葡萄糖滴定。 

视频: Biochemical Titration of Glycogen In vitro

Titration of Human Coronaviruses Using an Immunoperoxidase Assay

Determination of infectious viral titers is a basic and essential experimental approach for virologists. Classical plaque assays cannot be used for viruses that do not cause significant cytopathic effects, which is the case for prototype strains 229E and OC43 of human coronavirus (HCoV). Therefore, an alternative indirect immunoperoxidase assay (IPA) was developed for the detection and titration of these viruses and is described herein. Susceptible cells are inoculated with serial logarithmic dilutions of virus-containing samples in a 96-well plate format. After viral growth, viral detection by IPA yields the infectious virus titer, expressed as 'Tissue Culture Infectious Dose 50 percent' (TCID50). This represents the dilution of a virus-containing sample at which half of a series of laboratory wells contain infectious replicating virus. This technique provides a reliable method for the titration of HCoV-229E and HCoV-OC43 in biological samples such as cells, tissues and fluids. This article is based on work first reported in Methods in Molecular Biology (2008) volume 454, pages 93-102.

In this video, we demonstrate an alternative method for detection and titering of viruses using an enzymatic antigen detection technique known as an immunoperoxidase assay. Here, we will show you how to collect your viral samples, prepare the cells for testing, and finally the immunoperoxidase assay using serial dilutions to determine the viral titer.

人类冠状病毒的滴定使用免疫过氧化物酶含量

Determination of infectious viral titers is a basic and essential experimental approach for virologists. Classical plaque assays cannot be used for ...

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 ...

Collecting Variable-concentration Isothermal Titration Calorimetry Datasets in Order to Determine Binding Mechanisms

Isothermal titration calorimetry (ITC) is commonly used to determine the thermodynamic parameters associated with the binding of a ligand to a host macromolecule. ITC has some advantages over common spectroscopic approaches for studying host/ligand interactions. For example, the heat released or absorbed when the two components interact is directly measured and does not require any exogenous reporters. Thus the binding enthalpy and the association constant (Ka) are directly obtained from ITC data, and can be used to compute the entropic contribution. Moreover, the shape of the isotherm is dependent on the c-value and the mechanistic model involved. The c-value is defined as c = n[P]tKa, where [P]t is the protein concentration, and n is the number of ligand binding sites within the host. In many cases, multiple binding sites for a given ligand are non-equivalent and ITC allows the characterization of the thermodynamic binding parameters for each individual binding site. This however requires that the correct binding model be used. This choice can be problematic if different models can fit the same experimental data. We have previously shown that this problem can be circumvented by performing experiments at several c-values. The multiple isotherms obtained at different c-values are fit simultaneously to separate models. The correct model is next identified based on the goodness of fit across the entire variable-c dataset. This process is applied here to the aminoglycoside resistance-causing enzyme aminoglycoside N-6'-acetyltransferase-Ii (AAC(6')-Ii). Although our methodology is applicable to any system, the necessity of this strategy is better demonstrated with a macromolecule-ligand system showing allostery or cooperativity, and when different binding models provide essentially identical fits to the same data. To our knowledge, there are no such systems commercially available. AAC(6')-Ii, is a homo-dimer containing two active sites, showing cooperativity between the two subunits. However ITC data obtained at a single c-value can be fit equally well to at least two different models a two-sets-of-sites independent model and a two-site sequential (cooperative) model. Through varying the c-value as explained above, it was established that the correct binding model for AAC(6')-Ii is a two-site sequential binding model. Herein, we describe the steps that must be taken when performing ITC experiments in order to obtain datasets suitable for variable-c analyses.

ITC is a powerful tool for studying the binding of a ligand to its host. In complex systems however, several models may fit the data equally well. The method described here provides a means to elucidate the appropriate binding model for complex systems and extract the corresponding thermodynamic parameters.

收集可变浓度等温滴定量热数据集，以确定约束机制

Isothermal titration calorimetry (ITC) is commonly used to determine the thermodynamic parameters associated with the binding of a ligand to a host ...

Biochemical Reconstitution of Steroid Receptor&#x2022;Hsp90 Protein  Complexes and Reactivation of Ligand Binding

Hsp90 is an essential and highly abundant molecular chaperone protein that has been found to regulate more than 150 eukaryotic signaling proteins, including transcription factors (e.g. nuclear receptors, p53) and protein kinases (e.g. Src, Raf, Akt kinase) involved in cell cycling, tumorigenesis, apoptosis, and multiple eukaryotic signaling pathways 1,2. Of these many 'client' proteins for hsp90, the assembly of steroid receptor&bull;hsp90 complexes is the best defined (Figure 1). We present here an adaptable glucocorticoid receptor (GR) immunoprecipitation assay and in vitro GR&bull;hsp90 reconstitution method that may be readily used to probe eukaryotic hsp90 functional activity, hsp90-mediated steroid receptor ligand binding, and molecular chaperone cofactor requirements. For example, this assay can be used to test hsp90 cofactor requirements and the effects of adding exogenous compounds to the reconstitution process. 
The GR has been a particularly useful system for studying hsp90 because the receptor must be bound to hsp90 to have an open ligand binding cleft that is accessible to steroid 3. Endogenous, unliganded GR is present in the cytoplasm of mammalian cells noncovalently bound to hsp90. As found in the endogenous GR&bull;hsp90 heterocomplex, the GR ligand binding cleft is open and capable of binding steroid. If hsp90 dissociates from the GR or if its function is inhibited, the receptor is unable to bind steroid and requires reconstitution of the GR&bull;hsp90 heterocomplex before steroid binding activity is restored 4 . GR can be immunoprecipitated from cell cytosol using a monoclonal antibody, and proteins such as hsp90 complexed to the GR can be assayed by western blot. Steroid binding activity of the immunoprecipitated GR can be determined by incubating the immunopellet with [3H]steroid. 
Previous experiments have shown hsp90-mediated opening of the GR ligand binding cleft requires hsp70, a second molecular chaperone also essential for eukaryotic cell viability. Biochemical activity of hsp90 and hsp70 are catalyzed by co-chaperone proteins Hop, hsp40, and p23 5. A multiprotein chaperone machinery containing hsp90, hsp70, Hop, and hsp40 are endogenously present in eukaryotic cell cytoplasm, and reticulocyte lysate provides a chaperone-rich protein source 6. 
In the method presented, GR is immunoadsorbed from cell cytosol and stripped of the endogenous hsp90/hsp70 chaperone machinery using mild salt conditions. The salt-stripped GR is then incubated with reticulocyte lysate, ATP, and K+, which results in the reconstitution of the GR&bull;hsp90 heterocomplex and reactivation of steroid binding activity 7. This method can be utilized to test the effects of various chaperone cofactors, novel proteins, and experimental hsp90 or GR inhibitors in order to determine their functional significance on hsp90-mediated steroid binding 8-11.

An in vitro method for preparing functional glucocorticoid receptor (GR)&#x2022;hsp90 protein complexes from purified proteins and cellular lysates is described. The method utilizes immunoadsorption of recombinant GR followed by salt-stripping and protein complex reconstitution. The importance of cofactors and buffer conditions are discussed, as are potential method applications.

类固醇受体的生化重建•Hsp90的蛋白质复合物和配体激活绑定

Hsp90 is an essential and highly abundant molecular chaperone protein that has been found to regulate more than 150 eukaryotic signaling proteins, ...

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 ...

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the messagefor review see 1. However, it's now widely accepted that synonymous codon usage is the primary cause of non-uniform translational elongation rates1. Synonymous codons are not used with identical frequency. A bias exists in the use of synonymous codons with some codons used more frequently than others2. Codon bias is organism as well as tissue specific2,3. Moreover, frequency of codon usage is directly proportional to the concentrations of cognate tRNAs4. Thus, a frequently used codon will have higher multitude of corresponding tRNAs, which further implies that a frequent codon will be translated faster than an infrequent one. Thus, regions on mRNA enriched in rare codons (potential pause sites) will as a rule slow down ribosome movement on the message and cause accumulation of nascent peptides of the respective sizes5-8. These pause sites can have functional impact on the protein expression, mRNA stability and protein foldingfor review see 9. Indeed, it was shown that alleviation of such pause sites can alter ribosome movement on mRNA and subsequently may affect the efficiency of co-translational (in vivo) protein folding1,7,10,11. To understand the process of protein folding in vivo, in the cell, that is ultimately coupled to the process of protein synthesis it is essential to gain comprehensive insights into the impact of codon usage/tRNA content on the movement of ribosomes along mRNA during translational elongation.
Here we describe a simple technique that can be used to locate major translation pause sites for a given mRNA translated in various cell-free systems6-8. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA. The rationale is that at low-frequency codons, the increase in the residence time of the ribosomes results in increased amounts of nascent peptides of the corresponding sizes. In vitro transcribed mRNA is used for in vitro translational reactions in the presence of radioactively labeled amino acids to allow the detection of the nascent chains. In order to isolate ribosome bound nascent polypeptide complexes the translation reaction is layered on top of 30% glycerol solution followed by centrifugation. Nascent polypeptides in polysomal pellet are further treated with ribonuclease A and resolved by SDS PAGE. This technique can be potentially used for any protein and allows analysis of ribosome movement along mRNA and the detection of the major pause sites. Additionally, this protocol can be adapted to study factors and conditions that can alter ribosome movement and thus potentially can also alter the function/conformation of the protein.

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

A technique to identify translational pause sites on mRNA is described. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA, followed by the size analysis of the nascent chains using a denaturing gel electrophoresis.

核糖体束缚新生多肽的分离<em&gt;在体外</em&gt;确定沿mRNA的翻译暂停网站

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the ...

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 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.

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 ...

High-throughput Titration of Luciferase-expressing Recombinant Viruses

Standard plaque assays to determine infectious viral titers can be time consuming, are not amenable to a high volume of samples, and cannot be done with viruses that do not form plaques. As an alternative to plaque assays, we have developed a high-throughput titration method that allows for the simultaneous titration of a high volume of samples in a single day. This approach involves infection of the samples with a Firefly luciferase tagged virus, transfer of the infected samples onto an appropriate permissive cell line, subsequent addition of luciferin, reading of plates in order to obtain luminescence readings, and finally the conversion from luminescence to viral titers. The assessment of cytotoxicity using a metabolic viability dye can be easily incorporated in the workflow in parallel and provide valuable information in the context of a drug screen. This technique provides a reliable, high-throughput method to determine viral titers as an alternative to a standard plaque assay.

This article presents a high-throughput luciferase expression-based method of titrating various RNA and DNA viruses using automated and manual liquid handlers.

萤光素酶表达的重组病毒的高通量滴定

Standard plaque assays to determine infectious viral titers can be time consuming, are not amenable to a high volume of samples, and cannot be done with ...

Growth-based Determination and Biochemical Confirmation of Genetic Requirements for Protein Degradation in Saccharomyces cerevisiae

Regulated protein degradation is crucial for virtually every cellular function. Much of what is known about the molecular mechanisms and genetic requirements for eukaryotic protein degradation was initially established in Saccharomyces cerevisiae. Classical analyses of protein degradation have relied on biochemical pulse-chase and cycloheximide-chase methodologies. While these techniques provide sensitive means for observing protein degradation, they are laborious, time-consuming, and low-throughput. These approaches are not amenable to rapid or large-scale screening for mutations that prevent protein degradation. Here, a yeast growth-based assay for the facile identification of genetic requirements for protein degradation is described. In this assay, a reporter enzyme required for growth under specific selective conditions is fused to an unstable protein. Cells lacking the endogenous reporter enzyme but expressing the fusion protein can grow under selective conditions only when the fusion protein is stabilized (i.e. when protein degradation is compromised). In the growth assay described here, serial dilutions of wild-type and mutant yeast cells harboring a plasmid encoding a fusion protein are spotted onto selective and non-selective medium. Growth under selective conditions is consistent with degradation impairment by a given mutation. Increased protein abundance should be biochemically confirmed. A method for the rapid extraction of yeast proteins in a form suitable for electrophoresis and western blotting is also demonstrated. A growth-based readout for protein stability, combined with a simple protocol for protein extraction for biochemical analysis, facilitates rapid identification of genetic requirements for protein degradation. These techniques can be adapted to monitor degradation of a variety of short-lived proteins. In the example presented, the His3 enzyme, which is required for histidine biosynthesis, was fused to Deg1-Sec62. Deg1-Sec62 is targeted for degradation after it aberrantly engages the endoplasmic reticulum translocon. Cells harboring Deg1-Sec62-His3 were able to grow under selective conditions when the protein was stabilized.

Growth-based Determination and Biochemical Confirmation of Genetic Requirements for Protein Degradation in Saccharomyces cerevisiae

This article describes a yeast growth-based assay for the determination of genetic requirements for protein degradation. It also demonstrates a method for rapid extraction of yeast proteins, suitable for western blotting to biochemically confirm degradation requirements. These techniques can be adapted to monitor degradation of a variety of proteins.

糖原生化滴定

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