Name: quantification of coenzyme a in cells and tissues
Uploaded: 2019-09-27T14:00:00
Description: Watch this Scientific Journal Video about Quantification of Coenzyme A in Cells and Tissues at JoVE.com

作者承认，由BridgeBio LLC的子公司CoA治疗公司、国家卫生研究院拨款GM34496和美国黎巴嫩叙利亚联合慈善机构提供的赞助研究提供资金。

本议定书中提到的动物程序是按照第323和556号议定书进行的，并特别得到圣朱德儿童研究医院机构动物护理和使用委员会的批准。
1. 准备解决方案
注：使用超纯水用于所有解决方案，并在程序中注明。
<ol><li>在水中制备1 mM氢氧化钾（KOH）。</li>
	<li>在水中准备 0.25 M KOH。</li>
	<li>在水中准备 1 M Trizma-HCl 并调节到 pH 8.0。</li>
	<li>在醋酸酯（Optima级）中制?...

<ol>
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M., Leonardi, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overexpression+of+Nudt7+decreases+bile+acid+levels+and+peroxisomal+fatty+acid+oxidation+in+the+liver.">Overexpression of Nudt7 decreases bile acid levels and peroxisomal fatty acid oxidation in the liver.</a> Journal of Lipid Research. 60, 1005-1019 (2019).</li><li>Shumar, S. 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=Induction+of+neuron-specific+degradation+of+Coenzyme+A+models+pantothenate+kinase-associated+neurodegeneration+by+reducing+motor+coordination+in+mice.">Induction of neuron-specific degradation of Coenzyme A models pantothenate kinase-associated neurodegeneration by reducing motor coordination in mice.</a> PLoS ONE. 10, 0130013 (2015).</li><li>Zano, S. P., Pate, C., Frank, M., Rock, C. O., Jackowski, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correction+of+a+genetic+deficiency+in+pantothenate+kinase+1+using+phosphopantothenate+replacement+therapy.">Correction of a genetic deficiency in pantothenate kinase 1 using phosphopantothenate replacement therapy.</a> Molecular Genetics and Metabolism. 16, 281-288 (2015).</li><li>Sharma, L. K., 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=A+therapeutic+approach+to+pantothenate+kinase+associated+neurodegeneration.">A therapeutic approach to pantothenate kinase associated neurodegeneration.</a> Nature Communications. 9, 4399 (2018).</li><li>Alvarez-Cordoba, M., 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=Pantothenate+Rescues+Iron+Accumulation+in+Pantothenate+Kinase-Associated+Neurodegeneration+Depending+on+the+Type+of+Mutation.">Pantothenate Rescues Iron Accumulation in Pantothenate Kinase-Associated Neurodegeneration Depending on the Type of Mutation.</a> Molecular Neurobiology. 56, 3638-3656 (2019).</li><li>Arber, 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=iPSC-derived+neuronal+models+of+PANK2-associated+neurodegeneration+reveal+mitochondrial+dysfunction+contributing+to+early+disease.">iPSC-derived neuronal models of PANK2-associated neurodegeneration reveal mitochondrial dysfunction contributing to early disease.</a> PLoS One. 12, 0184104 (2017).</li><li>Di Meo, I., 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=Acetyl-4'-phosphopantetheine+is+stable+in+serum+and+prevents+phenotypes+induced+by+pantothenate+kinase+deficiency.">Acetyl-4'-phosphopantetheine is stable in serum and prevents phenotypes induced by pantothenate kinase deficiency.</a> Scientific Reports. 7, 11260 (2017).</li><li>Nishikawa, T., Edelstein, D., Brownlee, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+missing+link:+a+single+unifying+mechanism+for+diabetic+complications.">The missing link: a single unifying mechanism for diabetic complications.</a> Kidney International Supplements. 77, 26-30 (2000).</li><li>Tsuchiya, Y., Pham, U., Gout, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+measuring+CoA+and+CoA+derivatives+in+biological+samples.">Methods for measuring CoA and CoA derivatives in biological samples.</a> Biochemical Society Transactions. 42, 1107-1111 (2014).</li><li>Demoz, A., Netteland, B., Svardal, A., Mansoor, M. A., Berge, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Separation+and+Detection+of+Tissue+Coash+and+Long-Chain+Acyl-Coa+by+Reversed-Phase+High-Performance+Liquid-Chromatography+after+Precolumn+Derivatization+with+Monobromobimane.">Separation and Detection of Tissue Coash and Long-Chain Acyl-Coa by Reversed-Phase High-Performance Liquid-Chromatography after Precolumn Derivatization with Monobromobimane.</a> Journal of Chromatography. 635, 251-256 (1993).</li><li>Shimada, K., Mitamura, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Derivatization+of+Thiol-Containing+Compounds.">Derivatization of Thiol-Containing Compounds.</a> Journal of Chromatography B. 659, 227-241 (1994).</li><li>Minkler, P. E., Kerner, J., Ingalls, S. T., Hoppel, C. 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在这里，我们演示了一个可靠的、分步化的过程，用于量化细胞和动物组织中的总 CoA，具有广泛的线性检测功能，这些线性检测可在具有吸收性或荧光输出检测器的 HPLC 的实验室中进行。或者，质谱法是评估CoA和CoA硫化物的常用技术，但由于仪器的成本以及开发方法和解释数据所需的专业知识，因此不能广泛提供。适合用作质谱中自由 CoA 定量的内部标准的同位标记 CoA 不可用于商业上，并且仪?...

辅酶A（CoA）是所有生物中必不可少的合成物，由泛酸（也称为泛酸盐）或维生素B5合成。CoA是有机酸的主要细胞内载体，包括短链酸，如醋酸和琥珀酸，亚链酸，如丙酸和甲基甲酸酯，长链脂肪酸，如棕榈酸和油酸盐，超长链脂肪酸，如多不饱和脂肪酸，异种生物，如丙丙酸。有机酸与CoA酶形成硫酯联联系，使其在100多个中间代谢1反应中用作基质。CoA 硫化物也是基洛斯特调节剂和转录活化剂。现在，人们赞赏2，细胞总CoA供应是调节3，4;因此，CoA的可用性可以受到限制，CoA的缺陷可能是灾难性的，例如影响CoA生物合成5的遗传性遗传性疾病。泛酸激酶催化CoA生物合成的第一步（图1）和泛酸激酶相关神经退化，称为PKAN，是由PANK2基因6的突变引起的。CoA合成酶，由COASYN基因编码，催化CoA生物合成的最后两个步骤（图1）和COASY蛋白相关的神经退化，称为CoPAN，是由COASYN基因7的突变引起的。PKAN 和 CoPAN 都是遗传性神经退行性疾病，与大脑中的铁积累和 CoA 缺乏疾病病理学有关。
在组织8之间，总 CoA 的细胞水平各不相同，在各种生理、病理和药理状态下，总 CoA 可以增加或减少。肝CoA增加期间燃料从饲料切换到禁食状态9，肝CoA水平异常高的瘦素缺乏肥胖小鼠10。肝CoA减少对慢性乙醇摄入11。Pank2敲除小鼠模型中的脑CoA水平在围产期被抑制，但后来在成人阶段大脑CoA含量相当于野生型水平，表明在发育过程中有自适应CoA反应。通过转因或基因传递方法操作组织CoA含量会影响代谢和神经功能13、14、15。PKAN 或 CoPAN 潜在疗法的临床前开发包括细胞或组织 CoA 测量作为疗效指标 16、17、18、19、20.评估所有这些条件及其代谢或功能后果需要一种定量方法来测量总 CoA。
在许多实验室中，测量生物样品 CoA 的准确、可靠的检测是一项技术难题。不幸的是，没有探针可用于评估或量化在完整细胞中的CoA或CoA硫化物，虽然天然CoA硫化物的类比在利用酶21的CoA酯研究中被广泛用作机械探针。CoA 与游离磺酸 （-SH） 莫伊蒂一起转换为 CoA 硫化物（反之亦然）在转移到不同环境和细胞裂解期间在细胞或动物组织中快速。细胞中的大量丙基-CoA合成酶和丙基-CoA硫酯酶在CoA池内介导转化，利用CoA硫酯作为基质的其他酶在生物样品中保持活性，直到通过化学或物理淬火意味 着。通过丙烯转移酶将丙基组从CoA卸载到肉碱，是反应网络中可能改变CoA/CoA硫酯分布的一个例子。放射性示踪剂可用于测量细胞中的CoA合成率。目前测量生物样品中的CoA和CoA衍生物的方法已经审查了22种，包括耦合酶分光度测定测定、高压液相色谱和质谱法程序。然而，这些方法往往集中在特定的CoA分子物种上，并且对整个CoA池的变化视而不见。由于检测灵敏度低，并且线性范围有限，耦合酶测定通常需要大量输入材料。
我们的实验室已经开发出一种可靠的程序，用于定量培养细胞和动物组织中的总CoA。该战略包括所有CoA硫化物的水解，在样品制备过程中只产生免费CoA，而不是努力维持和分析CoA物种的整个谱系。该程序是单独公布的方法汇编，用于制备样品、CoA衍生、纯化和高压液相色谱 （HPLC） 后识别，以及通过吸光或荧光检测23，24，25。使用此程序获得的 CoA 测定使我们能够了解 CoA 法规，并开发治疗 CoA 缺陷的治疗方法。

<table>
	<tbody>
		<tr>
			<td>2-(2-pyridyl)-ethyl silica gel SPE column</td>
			<td>Millipore-Sigma</td>
			<td>54127-U</td>
			<td></td>
		</tr>
		<tr>
			<td>coenzyme A</td>
			<td>Avanti Polar Lipids</td>
			<td>870700</td>
			<td></td>
		</tr>
		<tr>
			<td>Gemini C18 3 &#956;m 100 &#197; HPLC column</td>
			<td>Phenomenex</td>
			<td>00F-4439-E0</td>
			<td></td>
		</tr>
		<tr>
			<td>monobromobimane</td>
			<td>ThermoFisher Scientific</td>
			<td>M-1378</td>
			<td></td>
		</tr>
		<tr>
			<td>Omni-Tip probe tissue disrupter</td>
			<td>Omni International</td>
			<td>32750H</td>
			<td></td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td>Fisher</td>
			<td>S37440</td>
			<td></td>
		</tr>
		<tr>
			<td>PowerGen 125 motorized rotor stator homogenizer</td>
			<td>ThermoFisher Scientific</td>
			<td>NC0530997</td>
			<td></td>
		</tr>
		<tr>
			<td>Spin-X centrifuge tube filter</td>
			<td>CoStar</td>
			<td>8161</td>
			<td></td>
		</tr>
		<tr>
			<td>Trizma-HCl</td>
			<td>Fisher</td>
			<td>T395-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Waters 2475 fluorescence detector</td>
			<td>Waters</td>
			<td>2475</td>
			<td></td>
		</tr>
		<tr>
			<td>Waters 2489 UV-Vis detector</td>
			<td>Waters</td>
			<td>2489</td>
			<td></td>
		</tr>
		<tr>
			<td>Waters e2695 separations module</td>
			<td>Waters</td>
			<td>e2695</td>
			<td></td>
		</tr>
	</tbody>
</table>

quantification of coenzyme a in cells and tissues

最新研究表明，细胞辅酶A（CoA）供应可能受到限制，对生长、新陈代谢和生存产生不利影响。细胞CoA的测量是一个挑战，由于其相对较低的丰度和自由CoA的动态转换到CoA硫化物，反过来，参与大量的代谢反应。介绍了一种方法，该方法在样品制备过程中通过潜在缺陷进行导航，以产生适用于许多生物医学实验室的广泛线性检测范围的检测。

利用mBBr将CoA的二醇衍生为荧光剂，然后利用反相HPLC纯化衍生的CoA-bimane，开发出一种相对快速、可靠的方法，用于检测培养细胞和组织的总CoA。首先生成标准曲线，其中已知且不断增加的 CoA-bimane 标准量单独注入，CoA-bimane 色谱图中峰值下的区域绘制为输入 CoA-bimane 的函数（图 4）。CoA-bimane 的吸收率最大值为 ±393 nM ，具有代表性的 HPLC 配置文件使用表 1中的洗?...

Watch this Scientific Journal Video about Quantification of Coenzyme A in Cells and Tissues at JoVE.com

Quantification of Coenzyme A in Cells and Tissues

该方法描述了来自培养细胞和动物组织的样品制备，在样品中提取和衍生辅酶A，然后高压液相色谱用于纯化和定量衍生辅酶A吸光或荧光检测。

细胞和组织中辅酶A的定量

Emerging research has revealed that the cellular coenzyme A (CoA) supply can become limiting with a detrimental impact on growth, metabolism and survival. ...

酶A，称为CoA，是细胞代谢的重要辅助因子。定量测量揭示了动物模型中营养和荷尔蒙状态的变化。此方法以简单可靠的方法量化细胞CoA的总量，包括CoA衍生物和自由CoA。脑铁积累的几种类型的神经退化与CoA生物合成途径基因的突变有关。这种方法可以应用于任何生物系统，因为所有生物体都需要CoA才能生存、生长和功能。首次研究者可能难以完成样品制备的第一步，其中重要的是保持样品绝对冷，然后迅速将它们转移到氢氧化钾。在转移到氢氧化钾之前，限制批次中样品的数量，并在使用实验样品之前练习转移。许多研究人员在准备生物样品以进行后期生化分析时，并不熟悉固相提取。首先，同时孵育三联培养皿，两个用于生化分析，一个用于确定可行的细胞数。例如，当培养接近后期亚氟密度时，人类 HepG2/C3A 细胞的密度为 6 到 8 倍 10 到 6 或 HEK 293T 细胞的密度约为 1.3 倍 10 至 7/100 毫米的培养皿，收获培养中的粘附细胞。然后，在同一文化菜中加入一毫升冰水。将细胞刮入盘中的冷水中，将细胞悬浮液转移到含有400微升的0.25-摩尔氢氧化钾和1.5毫升水的玻璃试管中，使pH值高于12。通过高旋转 10 秒，大力混合悬架。然后用石蜡膜紧紧覆盖，在55摄氏度下孵育一小时，在水浴中不摇晃。然后，加入160微升的一摩尔三氧化氢溶液和10微升100毫摩尔mBBr。通过高涡旋10秒混合。这使 pH 到大约 8，以支持带免费 CoA 的 mBBr 反应。用石蜡膜盖住管子，在室温下孵育样品两小时，使mBBr与CoA的硫醇发生反应。两小时后，加入100微升醋酸，通过涡流混合10秒钟，停止反应。管子中形成降水。接下来，以2000次g离心15分钟。要清除沉淀的细胞碎屑，请将上清液转移到玻璃试管中，用于固相提取柱清理。在开始分析之前，在玻璃试管中加入两毫升一毫升氢氧化钾，用于插入探针，用转子定子均质器进行组织破坏。将管子放在冰上直到使用。快速称量冷冻组织片，并记录每个样品的湿重。将组织转移到玻璃管中，使组织均质30秒。避免组织完全解冻。加入500微升0.25-摩尔氢氧化钾。漩涡在高10秒，并保持在冰上。这使高于 12 的样品的 pH 水解使 CoA 硫化剂产生总 CoA。用石蜡膜盖住管子。培养细胞的制备和组织是这个过程中最关键的步骤。必须快速添加高氢氧化钾，以防止CoA降解。在55摄氏度下在水浴中孵育样品两小时，不摇晃支持水解。然后，添加150微升的一摩尔三氧化氮和10微升100毫摩尔mBBr，使pH到约8，支持mBBr反应与免费的CoA。高涡高10秒。用石蜡膜盖住管子，在黑暗中在室温下孵育样品两小时，以确保所有CoA都用mBBr衍生。之后，加入100微升醋酸，并在高涡10秒内停止反应。在2000次g下离心15分钟，将沉淀的细胞碎屑颗粒，然后将上清液转移到玻璃试管中，用于固相提取柱清理。在室温下，用一毫升洗涤缓冲液平衡每一毫升一次性2-2-乙基乙基凝胶柱，以确保丙基功能组被质子化，并将作为阴安交换器发挥作用。然后，将样品上流水放在柱子上，然后收集水液。用一毫升的洗涤缓冲液清洗柱子两次，用一毫升水清洗一次，以清除任何未处理的物种。接下来，放入单独的玻璃试管中，用一毫升洗脱缓冲液清洗柱子两次，以收集CoA-bimane。在氮气下干燥管中的CoA-bimane样品。密封，完全覆盖管子，并储存在零下20摄氏度。准备好进行 HPLC 分析时，将样品重新在 300 微升水中重新暂停，然后通过高涡旋 10 秒进行大力混合。将重新沉淀的样品转移到离心管过滤器中，以5000次g离心10分钟，清除任何沉淀物。然后，将过滤过的样品转移到适合 HPLC 注射的玻璃瓶中。在这项研究中，一个具有代表性的 HPLC 配置文件显示了 C18 HPLC 列上 CoA-bimane 标准的保留时间。增加的CoA-bimane标准被单独注入，在CoA-bimane色谱的峰下的区域被绘制为输入CoA-bimane的函数。标准曲线反映了CoA-bimane在波长为393纳米时吸收的幅度。CoA-bimane的荧光也反映在393纳米的激发波长和470纳米的发射波长上。随后的 HPLC 分离和小鼠肝脏或人类培养的 C3A 细胞的典型检测配置文件在此以红色峰值表示，使用洗脱程序保留时间在 11 到 12 分钟之间。如果 mBBr 的反应不能产生可检测的结果，可能需要调整 Trizma-HCl 的量，以降低 pH 到大约 8。有可能检测更多的细胞分子，其中包含硫化物或硫酯的内饰作为双曼衍生物。例如，可以使用 HPLC 方法检测谷胱甘肽。这项技术将允许其他研究人员研究与代谢失衡相关的CoA功能障碍的潜在作用，如2型糖尿病的动物模型。研究人员在使用用于固相萃取的有机溶剂时，应使用个人防护设备。

quantification-of-coenzyme-a-in-cells-and-tissues

Research

JoVE Journal

Biochemistry

A Method for Measuring RNA N6-methyladenosine Modifications in Cells and Tissues

N6-Methyladenosine (m6A) modifications of RNA are diverse and ubiquitous amongst eukaryotes. They occur in mRNA, rRNA, tRNA, and microRNA. Recent studies have revealed that these reversible RNA modifications affect RNA splicing, translation, degradation, and localization. Multiple physiological processes, like circadian rhythms, stem cell pluripotency, fibrosis, triglyceride metabolism, and obesity are also controlled by m6A modifications. Immunoprecipitation/sequencing, mass spectrometry, and modified northern blotting are some of the methods commonly employed to measure m6A modifications. Herein, we present a northeastern blotting technique for measuring m6A modifications. The current protocol provides good size separation of RNA, better accommodation and standardization for various experimental designs, and clear delineation of m6A modifications in various sources of RNA. While m6A modifications are known to have a crucial impact on human physiology relating to circadian rhythms and obesity, their roles in other (patho)physiological states are unclear. Therefore, investigations on m6A modifications have immense possibility to provide key insights into molecular physiology.

A Method for Measuring RNA N6-methyladenosine Modifications in Cells and Tissues

A modified northern blotting method for measuring N6-methyladenosine (m6A) modifications in RNA is described. The current method can detect modifications in diverse RNAs and controls under various experimental designs.

一种测量方法RNA<em&gt;ñ</em<sup&gt; 6</sup&gt; -methyladenosine细胞和组织修改

N6-Methyladenosine (m6A) modifications of RNA are diverse and ubiquitous amongst eukaryotes. They occur in mRNA, rRNA, tRNA, and microRNA. Recent studies ...

科研

生物化学

Quantification of Bacterial Histidine Kinase Autophosphorylation Using a Nitrocellulose Binding Assay

We demonstrate a useful method for quantifying autophosphorylation of purified bacterial histidine kinases. Histidine kinases are known for their involvement in two-component signal transduction, a ubiquitous system through which bacteria sense and respond to environmental stimuli. Two-component signaling features autophosphorylation of a histidine kinase, followed by phosphotransfer to the receiver domain of a response regulator protein, which ultimately leads to an output response. Autophosphorylation of the histidine kinase is responsive to the presence of a cognate environmental stimulus, thereby giving bacteria a means to detect and respond to changes in the environment. Despite their importance in bacterial biology, histidine kinases remain poorly understood due to the inherent lability of phosphohistidine. Conventional methods for studying these proteins, such as SDS-PAGE autoradiography, have significant shortcomings. We have developed a nitrocellulose binding assay that can be used to characterize histidine kinases. The protocol for this assay is simple and easy to execute. Our method is higher throughput, less time-consuming, and offers a greater dynamic range than SDS-PAGE autoradiography.

We report and demonstrate an optimized nitrocellulose binding assay that can be used to quantify autophosphorylation of purified bacterial histidine kinases. Our method has several advantages over traditional SDS-PAGE based techniques, providing a valuable alternative for characterizing these important proteins.

使用硝酸纤维素结合分析细菌组氨酸激酶自身磷酸化的定量分析

We demonstrate a useful method for quantifying autophosphorylation of purified bacterial histidine kinases. Histidine kinases are known for their ...

Quantification of the Abundance and Charging Levels of Transfer RNAs in Escherichia coli

Transfer RNA (tRNA) is an essential part of the translational machinery in any organism. tRNAs bind and transfer amino acids to the translating ribosome. The relative levels of different tRNAs, and the ratio of aminoacylated tRNA to total tRNA, known as the charging level, are important factors in determining the accuracy and speed of translation. Therefore, the abundance and charging levels of tRNAs are important variables to measure when studying protein synthesis, for example under various stress conditions. Here, we describe a method for harvesting tRNA and directly measuring both the relative abundance and the absolute charging level of specific tRNA species in Escherichia coli. The tRNA is harvested in such a way that the labile bond between the tRNA and its amino acid is preserved. The RNA is then subjected to gel electrophoresis and Northern blotting, which results in separation of the charged and uncharged tRNAs. The levels of specific tRNAs in different samples can be compared due to the addition of spike-in cells for normalization. Prior to RNA purification, we add 5% of E. coli cells that overproduce the rare tRNAselC to each sample. The amount of the tRNA species of interest in a sample is then normalized to the amount of tRNAselC in the same sample. Addition of spike-in cells prior to RNA purification has the advantage over addition of purified spike-in RNAs that it also accounts for any differences in cell lysis efficiency between samples.

Quantification of the Abundance and Charging Levels of Transfer RNAs in Escherichia coli

Here we present a method for directly measuring transfer RNA charging levels from purified Escherichia coli RNA as well as a way to compare relative levels of transfer RNA, or any other short RNA, across different samples based on the addition of spike-in cells expressing a reference gene.

丰度和收费水平的转移 Rna 在大肠杆菌中的量化

Transfer RNA (tRNA) is an essential part of the translational machinery in any organism. tRNAs bind and transfer amino acids to the translating ribosome. ...

Quantification of Hypopigmentation Activity In Vitro

This study presents laboratory methods for the quantification of hypopigmentation activity in vitro. Melanin, the major pigment in melanocytes, is synthesized in response to multiple cellular and environmental factors. Melanin protects skin cells from ultraviolet damage, but also has biophysical and biochemical functions. Excessive production or accumulation of melanin in melanocytes can cause dermatological problems, such as freckles, dark spots, melasma, and moles. Therefore, the control of melanogenesis with hypopigmentation agents is important in individuals with clinical or cosmetic needs. Melanin is primarily synthesized in the melanosomes of melanocytes in a complex biochemical process called melanogenesis, which is influenced by extrinsic and intrinsic factors, such as hormones, inflammation, age, and ultraviolet light exposure. We describe three methods to determine the hypopigmentation activity of chemicals or natural substances in melanocytes: measurement of the 1) cellular tyrosinase activity and 2) melanin content, and 3) staining and quantifying cellular melanin with image analysis.
In melanogenesis, tyrosinase catalyzes the rate-limiting step that converts L-tyrosine into 3,4-dihydroxyphenylalanine (L-DOPA) and then into dopaquinone. Therefore, the inhibition of tyrosinase is a primary hypopigmentation mechanism. In cultured melanocytes, tyrosinase activity can be quantified by adding L-DOPA as a substrate and measuring dopaquinone production by spectrophotometry. Melanogenesis can also be measured by quantifying the melanin content. The melanin-containing cellular fraction is extracted with NaOH and melanin is quantified spectrophotometrically. Finally, the melanin content can be quantified by image analysis following Fontana-Masson staining of melanin. Although the results of these in vitro assays may not always be reproduced in human skin, these methods are widely used in melanogenesis research, especially as the initial step to identify potential hypopigmentation activity. These methods can also be used to assess melanocyte activity, growth, and differentiation. Consistent results with the three different methods ensure the validity of the effects.

We describe three experimental methods for evaluating the hypopigmentation activity of chemicals in vitro: quantification of 1) cellular tyrosinase activity and 2) melanin content, and 3) measurement of melanin by cellular melanin staining and image analysis.

低聚活性的体外定量

This study presents laboratory methods for the quantification of hypopigmentation activity in vitro. Melanin, the major pigment in melanocytes, is ...

Dissipative Microgravimetry to Study the Binding Dynamics of the Phospholipid Binding Protein Annexin A2 to Solid-supported Lipid Bilayers Using a Quartz Resonator

The dissipative quartz crystal microbalance technique is a simple and label-free approach to measure simultaneously the mass uptake and viscoelastic properties of the absorbed/immobilized mass on sensor surfaces, allowing the measurements of the interaction of proteins with solid-supported surfaces, such as lipid bilayers, in real-time and with a high sensitivity. Annexins are a highly conserved group of phospholipid-binding proteins that interact reversibly with the negatively charged headgroups via the coordination of calcium ions. Here, we describe a protocol that was employed to quantitatively analyze the binding of annexin A2 (AnxA2) to planar lipid bilayers prepared on the surface of a quartz sensor. This protocol is optimized to obtain robust and reproducible data and includes a detailed step-by-step description. The method can be applied to other membrane-binding proteins and bilayer compositions.

Here, we present an experimental protocol that can be employed to determine the binding affinities and mode of interaction of label-free phospholipid-binding protein annexin A2 with immobilized solid-supported bilayers (SLB) by simultaneously measuring the mass uptake and the viscoelastic properties of the protein annexin A2.

用石英谐振器研究磷脂结合蛋白附件 a2 与固体支持的脂质层结合动力学的耗散微重法

The dissipative quartz crystal microbalance technique is a simple and label-free approach to measure simultaneously the mass uptake and viscoelastic ...

Quantification of Protein Interaction Network Dynamics using Multiplexed Co-Immunoprecipitation

Dynamic protein-protein interactions control cellular behavior, from motility to DNA replication to signal transduction. However, monitoring dynamic interactions among multiple proteins in a protein interaction network is technically difficult. Here, we present a protocol for Quantitative Multiplex Immunoprecipitation (QMI), which allows quantitative assessment of fold changes in protein interactions based on relative fluorescence measurements of Proteins in Shared Complexes detected by Exposed Surface epitopes (PiSCES). In QMI, protein complexes from cell lysates are immunoprecipitated onto microspheres, and then probed with a labeled antibody for a different protein in order to quantify the abundance of PiSCES. Immunoprecipitation antibodies are conjugated to different MagBead spectral regions, which allows a flow cytometer to differentiate multiple parallel immunoprecipitations and simultaneously quantify the amount of probe antibody associated with each. QMI does not require genetic tagging and can be performed using minimal biomaterial compared to other immunoprecipitation methods. QMI can be adapted for any defined group of interacting proteins, and has thus far been used to characterize signaling networks in T cells and neuronal glutamate synapses. Results have led to new hypothesis generation with potential diagnostic and therapeutic applications. This protocol includes instructions to perform QMI, from the initial antibody panel selection through to running assays and analyzing data. The initial assembly of a QMI assay involves screening antibodies to generate a panel, and empirically determining an appropriate lysis buffer. The subsequent reagent preparation includes covalently coupling immunoprecipitation antibodies to MagBeads, and biotinylating probe antibodies so they can be labeled by a streptavidin-conjugated fluorophore. To run the assay, lysate is mixed with MagBeads overnight, and then beads are divided and incubated with different probe antibodies, and then a fluorophore label, and read by flow cytometry. Two statistical tests are performed to identify PiSCES that differ significantly between experimental conditions, and results are visualized using heatmaps or node-edge diagrams.

Quantitative Multiplex Immunoprecipitation (QMI) uses flow cytometry for sensitive detection of differences in the abundance of targeted protein-protein interactions between two samples. QMI can be performed using a small amount of biomaterial, does not require genetically engineered tags, and can be adapted for any previously defined protein interaction network.

使用多路复用共免疫沉淀对蛋白质相互作用网络动力学进行量化

Dynamic protein-protein interactions control cellular behavior, from motility to DNA replication to signal transduction. However, monitoring dynamic ...

Extracellular Glucose Depletion as an Indirect Measure of Glucose Uptake in Cells and Tissues Ex Vivo

The ongoing worldwide epidemic of diabetes increases the demand for the identification of environmental, nutritional, endocrine, genetic, and epigenetic factors affecting glucose uptake. The measurement of intracellular fluorescence is a widely used method to test the uptake of fluorescently-labeled glucose (FD-glucose) in cells in vitro, or for imaging glucose-consuming tissues in vivo. This assay assesses glucose uptake at a chosen time point. The intracellular analysis assumes that the metabolism of FD-glucose is slower than that of endogenous glucose, which participates in catabolic and anabolic reactions and signaling. However, dynamic glucose metabolism also alters uptake mechanisms, which would require kinetic measurements of glucose uptake in response to different factors. This article describes a method for measuring extracellular FD-glucose depletion and validates its correlation with intracellular FD-glucose uptake in cells and tissues ex vivo. Extracellular glucose depletion may be potentially applicable for high-throughput kinetic and dose-dependent studies, as well as identifying compounds with glycemic activity and their tissue-specific effects.

Extracellular Glucose Depletion as an Indirect Measure of Glucose Uptake in Cells and Tissues Ex Vivo

Extracellular depletion of fluorescently labeled glucose correlates with glucose uptake and could be used for high-throughput screening of glucose uptake in excised organs and cell cultures.

细胞外葡萄糖消耗作为细胞和组织离体葡萄糖摄取的间接测量

The ongoing worldwide epidemic of diabetes increases the demand for the identification of environmental, nutritional, endocrine, genetic, and epigenetic ...

mtDNA 合成和分布的高通量定量

High-Throughput Image-Based Quantification of Mitochondrial DNA Synthesis and Distribution

The vast majority of cellular processes require a continuous supply of energy, the most common carrier of which is the ATP molecule. Eukaryotic cells produce most of their ATP in the mitochondria by oxidative phosphorylation. Mitochondria are unique organelles because they have their own genome that is replicated and passed on to the next generation of cells. In contrast to the nuclear genome, there are multiple copies of the mitochondrial genome in the cell. The detailed study of the mechanisms responsible for the replication, repair, and maintenance of the mitochondrial genome is essential for understanding the proper functioning of mitochondria and whole cells under both normal and disease conditions. Here, a method that allows the high-throughput quantification of the synthesis and distribution of mitochondrial DNA (mtDNA) in human cells cultured in vitro is presented. This approach is based on the immunofluorescence detection of actively synthesized DNA molecules labeled by 5-bromo-2'-deoxyuridine (BrdU) incorporation and the concurrent detection of all the mtDNA molecules with anti-DNA antibodies. Additionally, the mitochondria are visualized with specific dyes or antibodies. The culturing of cells in a multi-well format and the utilization of an automated fluorescence microscope make it easier to study the dynamics of mtDNA and the morphology of mitochondria under a variety of experimental conditions in a relatively short time.

作者聚焦：基于图像的高通量线粒体 DNA 合成和分布定量  

A procedure for studying the dynamics of mitochondrial DNA (mtDNA) metabolism in cells using a multi-well plate format and automated immunofluorescence imaging to detect and quantify mtDNA synthesis and distribution is described. This can be further used to investigate the effects of various inhibitors, cellular stresses, and gene silencing on mtDNA metabolism.

基于图像的高通量线粒体DNA合成和分布定量

The vast majority of cellular processes require a continuous supply of energy, the most common carrier of which is the ATP molecule. Eukaryotic cells ...