一NanoDrop 2000C分光光度计 NanoDrop技术是基于一个创新的样品保留系统使用的表面张力，保持和测量两个光学基座之间没有使用试管或毛细血管微量样品。 NanoDrop 2000C分光光度计使用这项技术，快速，方便地测量0.5-2μL，液滴的蛋白质，DNA，RNA，和其他生物大分子。这种能力已变得越来越重要的分子生物学技术的不断发展，使用数额较小的材料进行分析。在样本有限的条件下的微量分光光度计的理想选择。然而，易于使用，快速测量周期，和广泛的浓度范围内，也使分光光度计适合当充足的样本量。测量周期也大大降低，帮助科学家提高其整个工作流程的效率。 microsample是直接放置在检测表面的顶部和表面张力的光纤的两端之间创建一个液柱。这液柱形成了一个垂直的光路。一个氙闪光灯提供光源和光谱仪采用线性CCD阵列是用来分析光通过样品传递。 从系统中删除传统的遏制设备，如试管，有以下几个优点：测量所需要的极少量的样品，清理只需要实验室擦拭抹光表面，可以实时改变路径的长度测量过程中。 NanoDrop 2000C自动确定最优路径长度（1毫米至0.05毫米），未经稀释提供最广泛的可能的蛋白浓度测量。通过缩短路径长度，较高浓度的蛋白质可以衡量的。这有效地消除了需要执行的大多数蛋白质样品稀释。例如，NanoDrop 2000C可以测量BSA的浓度高达400毫克/毫升。 NanoDrop 2000C分光光度计是一种全方位的DNA，RNA，蛋白质和其他生物大分子的吸光度测量分光光度计。这个视频协议，将侧重于测量的蛋白质。 二。微量法测定蛋白浓度使用A280吸光度测量 A. A280测量原理蛋白质A280方法是适用于纯化的蛋白质含有色氨酸，酪氨酸，苯丙氨酸残基或半胱氨酸半胱氨酸二硫键和展览在280 nm处的吸光度。这种方法使用的质量消光系数，摩尔消光系数A280吸光度值，计算纯化的蛋白浓度组合。直接A280测量的优点是不需要标准曲线的生成，以确定蛋白质浓度。如果样品是一个未知的蛋白质溶液，细胞裂解液，或粗蛋白提取物，然后使用预先配置的比色方法，如NanoDrop 2000/2000c BCA，皮尔斯660纳米，布拉德福德和洛瑞检测之一，是建议。 B.微量蛋白质A280测量 - 启动<ol><li>选择从主菜单中的蛋白质A280应用程序。 </li><li>选择从下拉列表中测量样品类型。默认设置为推荐未知蛋白质的混合​​物，其中1 ABS = 1毫克/毫升。如果测量先前特点的纯化蛋白质，然后大规模灭绝系数或摩尔消光系数和分子量可进入更精确地确定蛋白浓度。 </li><li>从下拉列表中选择浓度单位。 </li></ol> C.微量蛋白质A280测量 - 消隐<ol><li>使用适当的缓冲区，建立一个空白。重要的是要使用其中的蛋白质暂停缓冲。使用缓冲区应该是相同的的pH值和一个类似的样品溶液的离子强度。注：RIPA缓冲区贡献太多吸光度在280 nm，因此不符合直接A280蛋白质测量。悬浮在RIPA缓冲液的蛋白质，它是建议使用一个兼容RIPA缓冲液比色法测定蛋白浓度。 </li><li>加入2μL的底部基座上，适当的消隐的解决方案，降低了手臂，然后单击空白。 </li><li>擦拭从空白的最低和最高使用干燥的实验室擦拭基座解决方案。 </li></ol> D.微量蛋白质A280三围尺寸重要注意事项：同质化的样本是极其因为这种体积小，重要的是被测量。为了确保样品均匀，轻轻地，彻底混合样品前测量等分。混合和移液时，避免引入气泡。 由于在不同的蛋白质之间的表面张力变异，我们建议加载2μL的样品，以确保形成正确的列。始终使用低保留枪头。要加载一个样本，同时驱逐的解决方案，以防止从坚持枪头外解决方案，触摸枪头，以较低的光学基座表面。比样品量，样品中的气泡，防止井喷和引进排出少。 <ol><li>在相应字段中输入一个样品编号，负载2μL为空白，然后单击措施的第一个样本。一个新鲜的样品2μL等分应当用于每次测量。 </li><li>取出的最低和最高使用干燥的实验室擦拭基座样品。 </li></ol> E.微量蛋白质A280测量清洁一个普通的，不掉毛，实验室擦拭往往是足够的清洁光学测量之间的底座。 F.使蛋白质A280测量翻新含有表面活性剂的解决方案和试剂，可能会uncondition随着时间的推移测量底座的表面，防止样品液柱形成。扁平化是一个较低的底座上的液滴成为无条件的光学表面的指标。如果表面性质已被泄露，翻新基座重要的是要确保样品列形成。如果发生这种情况，BUFF的大力使用实验室擦拭或使用NanoDrop立柱检修复合（PR - 1）的指示的光学表面。 三。微量法测定蛋白浓度使用比色法 A.比色法检测原理 NanoDrop 2000C分光光度计也可以用来测量未知的蛋白质溶液，细胞裂解液，粗蛋白提取物，采用比色法。 色度的方法是间接的方法，包括与样品的蛋白质成分的染料能吸收光线在可见光波长范围内产生一个新的复杂的相互作用。 NanoDrop 2000C分光光度计有几个预配置，包括基本能力评估，，布拉德福德，皮尔斯660和Lowry方法比色法。 BCA法测定将展示使用微量分光光度计比色法检测作为一个例子。 （截图） BCA（Bicinchoninic酸）法是一种常见的比色法通常用于稀的蛋白质溶液和蛋白质中存在的组件，具有显着的紫外线（280纳米）的吸光度。不同于蛋白质的A280方法，蛋白质的基本能力评估方法的要求，之前生成，可以测量样品中蛋白质浓度的标准曲线。 该方法采用的检测试剂bicinchoninic酸（BCA）。测量562纳米铜BCA螯合形成的蛋白质的存在，并在750 nm的正常化。 B.微量BCA法测定测量BCA含量的制备<ol><li>所需的试剂都包含在还原剂兼容工具包（Thermo Scientific的皮尔斯猫没有23250。）：BCA试剂，BCA试剂B，兼容性试剂，重组缓冲区，和白蛋白（BSA）标准。 </li><li>所有未知的蛋白质和蛋白质的标准平衡至室温，调匀。 </li><li>准备足够的标准和要测量的样品使用的试剂50:1的比例一个新的工作试剂：乙</li><li>对于微检测，使用工作试剂按1:1的比例进行采样。加入10μL的工作有足够的管试剂每管以涵盖所有样品和标准。对于高音域的检测，工作试剂的使用20:1的比例进行采样。加入190μL的工作有足够的管试剂每管以涵盖所有样品和标准。皮尔斯BCA试剂盒文学，以确定其中的比例是适合您的样品。 </li><li>含试剂每管加入10μL标准品或样品。充分混匀，轻轻振荡。 </li><li>管在37 ° C下30分钟。 </li><li>允许的反应，以平衡至室温（约10分钟）。 </li></ol> C.微量BCA法测定测量生成标准曲线<ol><li>选择从主菜单中的蛋白质的基本能力评估方法。如果波长出现验证窗口，确保手臂下来。 </li><li>在右窗格中的表的标准浓度为每输入值。该软件允许参考和高达7 additional标准。应在重复测量基准和/或标准。 注：标准曲线生成的最低要求是两个标准的测量或零的参考，并至少有一个标准的测量。这是建议，包括必要的覆盖预期的实验浓度范围内的其他标准。 </li><li>建立一个空白。比色法空白一般用去离子H 2 O。 </li><li>加入2μL生署2，将底部基座，较低的手臂，然后单击空白。只有一个空白是必要的职权范围和标准的所有后续测量。 </li><li>建立吸取2微升等份，含蛋白质没有到较低的底座只工作试剂和缓冲的参考。下手臂，然后单击测量。 </li><li>规定的标准“选项卡上，突出显示所需的标准所需的较低的底座上的标准，并加入2微升。下手臂，然后单击测量。重复所有的标准的过程中。请务必来衡量一切的标准测量样品之前。要查看标准曲线，单击“查看标准曲线。 </li></ol> D.微量BCA法测定测量2μL，蛋白质测量<ol><li>已测的所有标准后，点击“样本”按钮。输入样品编号。加载到较低的底座2μL的样品，并单击“测量”。一个新鲜的2μL的样品分装，应使用每个测量。 </li><li>用的最低和最高使用干燥的实验室擦拭基座样品。 </li></ol> E.微量BCA法测定测量清洗和翻新进行清洗和翻新直接A280吸光度测量。 代表性的成果： 微量法测定蛋白浓度是由一个直接的A280测量或间接比色法。 A280测量例如决定对感兴趣的蛋白质的消光系数为基础的蛋白浓度。 BCA比色法示例确定一个已知的蛋白质浓度的标准曲线为基础的蛋白浓度。

<ol>
	<li>Aitken, A., Learmonth, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+determination+by+UV+absorption.">Protein determination by UV absorption.</a> , (1996).</li><li>Bradford, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+and+sensitive+method+for+the+quantitation+of+microgram+quantities+of+protein+utilizing+the+principle+of+protein-dye+binding.">A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.</a> Anal. Biochem. 72, 248-254 (1976).</li><li>Lowry, O. H., Rosebrough, N. J., Farr, A., Randall, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+measurement+with+the+Folin+phenol+reagent.">Protein measurement with the Folin phenol reagent.</a> J. Biol. Chem. 193, 265-275 (1951).</li><li>Reisner, A. H., Nemes, P., Bucholtz, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+Coomassie+Brilliant+Blue+G250+perchloric+acid+solution+for+staining+in+electrophoresis+and+isoelectric+focusing+on+polyacrilamide+gels.">Use of Coomassie Brilliant Blue G250 perchloric acid solution for staining in electrophoresis and isoelectric focusing on polyacrilamide gels.</a> Anal. Biochem. 64, 509-516 (1975).</li><li>Simonian, M. H., Smith, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spectrophotometric+and+colorimetric+determination+of+protein+concentration.">Spectrophotometric and colorimetric determination of protein concentration.</a> Curr. Protoc. Mol. Biol. 10:1A.17. , (2006).</li><li>Smith, P. K., Krohn, R. I., Hermanson, G. T., Mallia, A. K., Gartner, F. H., Provenzano, F. u. j. i. m. o. t. o., Goeke, E. K., Olson, N. M., J, B., Klenk, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+protein+using+bicinchoninic+acid.">Measurement of protein using bicinchoninic acid.</a> Anal. Biochem. 150, 76-85 (1985).</li><li>Stoscheck, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitation+of+protein.">Quantitation of protein.</a> Meth. Enzymol. 182, 50-68 (1990).</li></ol>

作者是赛默飞世尔科技产生本文中使用的试剂和工具的员工。

微量定量使用一个样品的内在的表面张力特性，形成了两种测量表面之间的液柱。遏制设备的情况下，允许实时路径的长度来改变，根本上消除了需要进行稀释。这种能力显着提高蛋白浓度的动态范围以及测量速度。通过使用极少量的样品，大量的样品，可以快速，准确地分析，使科学家能够采取更多的测量，并实现更好的质量控制。此外，如果必要的话，宝贵的样品可以测量后收回。这样的小卷正在测量时，样品的均匀性是极为重要的的，以避免抽样误差。低保留枪头应始终被用于装载样本之间的样品复制，提示应改为。基座表面必须正确清洁和空调，以确保最准确的结果。最后，微量分光光度计是理想然而，当样品是有限的，易于使用，速度，和广泛的光谱仪的动态范围，使其适合时，样品是丰富的。

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microvolume protein concentration determination using the nanodrop 2000c spectrophotometer

传统的分光需要样品放入试管或毛细血管。这往往是不切实际的，由于有限的样本通常用于蛋白质分析的卷的。 Thermo Scientific的NanoDrop 2000C分光光度计解决这个问题与创新的样品保留系统，拥有两个使用液体的表面张力特性测量表面的微量样品，使卷在低0.5-2μL的样品定量。消除试管或毛细血管允许在实时路径的长度变化，从而降低了测量时间，同时大大提高了动态范围，可以测量蛋白质浓度。需要稀释，也消除了，并准备样品定量测量表面可以简单的实验室擦拭擦拭相对容易。此视频文章提出修改传统的蛋白浓度测定方法微量蛋白质的金额使用A280吸光度读数或BCA比色法定量。

Watch this Scientific Journal Video about Microvolume Protein Concentration Determination using the NanoDrop 2000c Spectrophotometer at JoVE.com

Microvolume Protein Concentration Determination using the NanoDrop 2000c Spectrophotometer

微量样品定量分光光度计系统，采用天然的表面张力，无需使用比色皿或毛细血管保留样本。用这种方法的动态范围，通过它们可以测量蛋白质浓度和速度都大大增加。

微量法测定蛋白浓度使用NanoDrop 2000C分光光度计

Traditional spectrophotometry requires placing samples into cuvettes or capillaries. This is often impractical due to the limited sample volumes often ...

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microvolume-protein-concentration-determination-using-nanodrop-2000c

Research

JoVE Journal

Biology

113.8K 次观看.  Thermo Scientific NanoDrop Products. 中文Deutsch Français Español 日本語 

视频: Microvolume Protein Concentration Determination using the NanoDrop 2000c Spectrophotometer

Crystallizing Membrane Proteins for Structure Determination using Lipidic Mesophases

A detailed protocol for crystallizing membrane proteins by using lipidic mesophases is described. This method has variously been referred to as the lipidic cubic phase or in meso method. The method has been shown to be quite versatile in that it has been used to solve X-ray crystallographic structures of prokaryotic and eukaryotic proteins, proteins that are monomeric, homo- and hetero-multimeric, chromophore-containing and chromophore-free, and alpha-helical and beta-barrel proteins. Recent successes using in meso crystallization are the human engineered beta2-adrenergic and adenosine A2a G protein-coupled receptors. Protocols are presented for reconstituting the membrane protein into the monoolein-based mesophase, and for setting up crystallizations in the manual mode. Additional steps in the overall process, such as crystal harvesting, are to be addressed in future video articles. The time required to prepare the protein-loaded mesophase and to set up a crystallization plate manually is about one hour.

Herein is described the procedure implemented in the Caffrey Membrane Structural and Functional Biology Group to set up manually crystallization trials of membrane proteins in lipidic mesophases.

使用油脂介孔结构的测定结晶膜蛋白

A detailed protocol for crystallizing membrane proteins by using lipidic mesophases is described. This method has variously been referred to as the ...

科研

生物学

The Importance of Correct Protein Concentration for Kinetics and Affinity Determination in Structure-function Analysis 

In this study, we explore the interaction between the bovine cysteine protease inhibitor cystatin B and a catalytically inactive form of papain (Fig. 1), a plant cysteine protease, by real-time label-free analysis using Biacore X100. Several cystatin B variants with point mutations in areas of interaction with papain, are produced. For each cystatin B variant we determine its specific binding concentration using calibration-free concentration analysis (CFCA) and compare the values obtained with total protein concentration as determined by A280. After that, the kinetics of each cystatin B variant binding to papain is measured using single-cycle kinetics (SCK). We show that one of the four cystatin B variants we examine is only partially active for binding. This partial activity, revealed by CFCA, translates to a significant difference in the association rate constant (ka) and affinity (KD), compared to the values calculated using total protein concentration. Using CFCA in combination with kinetic analysis in a structure-function study contributes to obtaining reliable results, and helps to make the right interpretation of the interaction mechanism.

The Importance of Correct Protein Concentration for Kinetics and Affinity Determination in Structure-function Analysis

We apply label-free protein interaction analysis using Biacore X100 for structure-function analysis of the binding of several cystatin B mutants to papain through kinetic characterization. Calibration-free concentration analysis (CFCA) measures the concentration of protein with retained binding activity without the need for a standard curve. We show that confirmation of concentrations using CFCA increases the reliability of the kinetic analysis and that kinetic constants can reliably be determined even if the activity of a recombinant protein is reduced.

正确的蛋白浓度的动力学结构与功能分析和亲和力测定的重要性

In this study, we explore the interaction between the bovine cysteine protease inhibitor cystatin B and a catalytically inactive form of papain (Fig. 1), ...

Using the GELFREE 8100 Fractionation System for Molecular Weight-Based Fractionation with Liquid Phase Recovery

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based fractionation. The system is comprised of single-use, 8-sample capacity cartridges and a benchtop GELFREE Fractionation Instrument. During separation, a constant voltage is applied between the anode and cathode reservoirs, and each protein mixture is electrophoretically driven from a loading chamber into a specially designed gel column gel. Proteins are concentrated into a tight band in a stacking gel, and separated based on their respective electrophoretic mobilities in a resolving gel. As proteins elute from the column, they are trapped and concentrated in liquid phase in the collection chamber, free of the gel. The instrument is then paused at specific time intervals, and fractions are collected using a pipette. This process is repeated until all desired fractions have been collected. If fewer than 8 samples are run on a cartridge, any unused chambers can be used in subsequent separations.
This novel technology facilitates the quick and simple separation of up to 8 complex protein mixtures simultaneously, and offers several advantages when compared to previously available fractionation methods. This system is capable of fractionating up to 1mg of total protein per channel, for a total of 8mg per cartridge. Intact proteins over a broad mass range are separated on the basis of molecular weight, retaining important physiochemical properties of the analyte. The liquid phase entrapment provides for high recovery while eliminating the need for band or spot cutting, making the fractionation process highly reproducible1.

The accompanying video describes the use of the GELFREE 8100 Fractionation System, which partitions complex protein samples on the basis of molecular weight and recovers the fractions in liquid phase. The video describes how the technology works, how it is used, and provides resultant data, with polyacrylamide gel electrophoresis analysis of fractionated bovine liver homogenate.

液相恢复GELFREE 8100分馏系统使用基于分子量分馏

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based ...

Analysis of the Development of a Morphological Phenotype as a Function of Protein Concentration in Budding Yeast

Gene deletion and protein overexpression are common methods for studying functions of proteins. In this article, we describe a protocol for analysis of phenotype development as a function of protein concentration at population and single-cell levels in Saccharomyces cerevisiae. Although this protocol is based on the overexpression of a protein, it can easily be adapted for morphological phenotypes dependent on suppression of protein expression. Our lab is interested in studying the signaling properties of the endocytic adaptor protein epsin. To that purpose we used a dominant negative approach in which we over-expressed the conserved Epsin N-Terminal Homology (ENTH) domain in order to interfere with the functions of endogenous epsin-2 (Ent2 or YLR206W). We observed that overexpression of the ENTH domain of Ent2 (ENTH2) in wild type cells led to a cell division defect that is dependent on the mislocalization of a family of scaffolding proteins, septins.

Gene deletion and protein overexpression are common methods for studying functions of proteins. In this article, we describe a protocol for analysis of phenotype development as a function of protein concentration at population and single-cell levels in Saccharomyces cerevisiae.

在芽殖酵母中的蛋白浓度的功能形态学表型的发展分析

Gene deletion and protein overexpression are common methods for studying functions of proteins. In this article, we describe a protocol for analysis of ...

Tissue Determination Using the Animal Cap Transplant (ACT) Assay in Xenopus laevis

Many proteins play a dual role in embryonic development. Those that regulate cell fate determination in a specific tissue can also affect the development of a larger region of the embryo. This makes defining its role in a particular tissue difficult to analyze. For example, noggin overexpression in Xenopus laevis embryos causes the expansion of the entire anterior region, including the eye1,2. From this result, it is not known if Noggin plays a direct role in eye determination or that by causing an expansion of neural tissue, Noggin indirectly affects eye formation. Having this complex phenotype makes studying its eye-specific role in cell fate determination difficult to analyze. We have developed an assay that overcomes this problem. Taking advantage of the pluripotent nature of the Xenopus laevis animal cap 3, we have developed an assay to test the ability of gene product(s), like noggin or the eye field transcription factors (EFTFs), to transform caps into particular tissue or cell types by transplanting this tissue onto the side of the embryo 4. While we have found either Noggin protein treatment or a collection of transcription factors can determine retinal cell fate in animal caps, this procedure could be used to identify gene product(s) involved in specifying other tissues as well.

Tissue Determination Using the Animal Cap Transplant ACT Assay in Xenopus laevis

Animal caps overexpressing gene product(s) are transplanted to the flank of developing Xenopus laevis embryos in order to establish whether tissue is determined.

组织测定动物第移植（ACT）的含量非洲爪蟾</em

Many proteins play a dual role in embryonic development. Those that regulate cell fate determination in a specific tissue can also affect the development ...

Quantification of dsDNA using the Hitachi F-7000 Fluorescence Spectrophotometer and PicoGreen Dye

Quantification of DNA, especially in small concentrations, is an important task with a wide range of biological applications including standard molecular biology assays such as synthesis and purification of DNA, diagnostic applications such as quantification of DNA amplification products, and detection of DNA molecules in drug preparations. During this video we will demonstrate the capability of the Hitachi F-7000 Fluorescence Spectrophotometer equipped with a Micro Plate Reader accessory to perform dsDNA quantification using Molecular Probes Quant-it PicoGreen dye reagent kit.
The F-7000 Fluorescence Spectrophotometer offers high sensitivity and high speed measurements. It is a highly flexible system capable of measuring fluorescence, luminescence, and phosphorescence. Several measuring modes are available, including wavelength scan, time scan, photometry and 3-D scan measurement. The spectrophotometer has sensitivity in the range of 50 picomoles of fluorescein when using a 300 &#956;L sample volume in the microplate, and is capable of measuring scan speeds of 60,000 nm/minute. It also has a wide dynamic range of up to 5 orders of magnitude which allows for the use of calibration curves over a wide range of concentrations. The optical system uses all reflective optics for maximum energy and sensitivity. The standard wavelength range is 200 to 750 nm, and can be extended to 900 nm when using one of the optional near infrared photomultipliers. The system allows optional temperature control for the plate reader from 5 to 60 degrees Celsius using an optional external temperature controlled liquid circulator. The microplate reader allows for the use of 96 well microplates, and the measuring speed for 96 wells is less than 60 seconds when using the kinetics mode.
Software controls for the F-7000 and Microplate Reader are also highly flexible. Samples may be set in either column or row formats, and any combination of wells may be chosen for sample measurements. This allows for optimal utilization of the microplate. Additionally, the software allows importing micro plate sample configurations created in Excel and saved in comma separated values, or "csv" format. Microplate measuring configurations can be saved and recalled by the software for convenience and increased productivity. Data results can be output to a standard report, to Excel, or to an optional Report Generator Program.

Demonstration of quantification of dsDNA using Molecular Probes PicoGreen dye and Hitachi F-7000 Fluorescence Spectrophotometer equipped with a microplate reader accessory.

双链DNA定量，使用日立F - 7000荧光分光光度计和PicoGreen染料

Quantification of DNA, especially in small concentrations, is an important task with a wide range of biological applications including standard molecular ...

NanoDrop Microvolume Quantitation of Nucleic Acids

Biomolecular assays are continually being developed that use progressively smaller amounts of material, often precluding the use of conventional cuvette-based instruments for nucleic acid quantitation for those that can perform microvolume quantitation.
The NanoDrop microvolume sample retention system (Thermo Scientific NanoDrop Products) functions by combining fiber optic technology and natural surface tension properties to capture and retain minute amounts of sample independent of traditional containment apparatus such as cuvettes or capillaries. Furthermore, the system employs shorter path lengths, which result in a broad range of nucleic acid concentration measurements, essentially eliminating the need to perform dilutions. Reducing the volume of sample required for spectroscopic analysis also facilitates the inclusion of additional quality control steps throughout many molecular workflows, increasing efficiency and ultimately leading to greater confidence in downstream results.
The need for high-sensitivity fluorescent analysis of limited mass has also emerged with recent experimental advances. Using the same microvolume sample retention technology, fluorescent measurements may be performed with 2 &mu;L of material, allowing fluorescent assays volume requirements to be significantly reduced. Such microreactions of 10 &mu;L or less are now possible using a dedicated microvolume fluorospectrometer.
Two microvolume nucleic acid quantitation protocols will be demonstrated that use integrated sample retention systems as practical alternatives to traditional cuvette-based protocols. First, a direct A260 absorbance method using a microvolume spectrophotometer is described. This is followed by a demonstration of a fluorescence-based method that enables reduced-volume fluorescence reactions with a microvolume fluorospectrometer. These novel techniques enable the assessment of nucleic acid concentrations ranging from 1 pg/ &mu;L to 15,000 ng/ &mu;L with minimal consumption of sample.

The use of NanoDrop microvolume systems as practical and efficient alternatives to traditional nucleic acid quantitation methodology is described through the demonstration of two microvolume nucleic acid quantitation protocols.

NanoDrop微量核酸的定量

Biomolecular assays are continually being developed that use progressively smaller amounts of material, often precluding the use of conventional ...

Concentration Determination of Nucleic Acids and Proteins Using the Micro-volume Bio-spec Nano Spectrophotometer

Nucleic Acid quantitation procedures have advanced significantly in the last three decades. More and more, molecular biologists require consistent small-volume analysis of nucleic acid samples for their experiments. The BioSpec-nano provides a potential solution to the problems of inaccurate, non-reproducible results, inherent in current DNA quantitation methods, via specialized optics and a sensitive PDA detector. The BioSpec-nano also has automated functionality such that mounting, measurement, and cleaning are done by the instrument, thereby eliminating tedious, repetitive, and inconsistent placement of the fiber optic element and manual cleaning.
In this study, data is presented on the quantification of DNA and protein, as well as on measurement reproducibility and accuracy. Automated sample contact and rapid scanning allows measurement in three seconds, resulting in excellent throughput. Data analysis is carried out using the built-in features of the software. The formula used for calculating DNA concentration is:
Sample Concentration = DF &middot; (OD260-OD320)&middot; NACF (1)
Where DF = sample dilution factor and NACF = nucleic acid concentration factor.
The Nucleic Acid concentration factor is set in accordance with the analyte selected1.
Protein concentration results can be expressed as &mu;g/ mL or as moles/L by entering e280 and molecular weight values respectively. When residue values for Tyr, Trp and Cysteine (S-S bond) are entered in the e280Calc tab, the extinction coefficient values are calculated as e280 = 5500 x (Trp residues) + 1490 x (Tyr residues) + 125 x (cysteine S-S bond). The e280 value is used by the software for concentration calculation.
In addition to concentration determination of nucleic acids and protein, the BioSpec-nano can be used as an ultra micro-volume spectrophotometer for many other analytes or as a standard spectrophotometer using 5 mm pathlength cells.

This communication presents data on the accuracy and reproducibility of the BioSpec-nano UV-VIS spectrophotometer for dsDNA and protein quantitation. Even with ultra-small volumes (1 to 2 L), reproducibility is excellent, while the automated wiping function improves throughput and results in minimal carryover for more precise results.

使用微量的生物规格的纳米分光光度计核酸和蛋白的浓度测定

Nucleic Acid quantitation procedures have advanced significantly in the last three decades.  More and more, molecular biologists require consistent ...

Protein Purification-free Method of Binding Affinity Determination by Microscale Thermophoresis

Quantitative characterization of protein interactions is essential in practically any field of life sciences, particularly drug discovery. Most of currently available methods of KD determination require access to purified protein of interest, generation of which can be time-consuming and expensive. We have developed a protocol that allows for determination of binding affinity by microscale thermophoresis (MST) without purification of the target protein from cell lysates. The method involves overexpression of the GFP-fused protein and cell lysis in non-denaturing conditions. Application of the method to STAT3-GFP transiently expressed in HEK293 cells allowed to determine for the first time the affinity of the well-studied transcription factor to oligonucleotides with different sequences. The protocol is straightforward and can have a variety of application for studying interactions of proteins with small molecules, peptides, DNA, RNA, and proteins.

Microscale thermophoresis (MST) can be widely used for determination of binding affinity without purification of the target protein from cell lysates. The protocol involves overexpression of the GFP-fused protein, cell lysis in non-denaturing conditions, and detection of MST signal in the presence of varying concentrations of the ligand.

蛋白质纯化方法测定结合亲和力微尺度热泳

Quantitative characterization of protein  interactions is essential in practically any field of life sciences,  particularly drug discovery. Most of ...

Determination of Protein-ligand Interactions Using Differential Scanning Fluorimetry

A wide range of methods are currently available for determining the dissociation constant between a protein and interacting small molecules. However, most of these require access to specialist equipment, and often require a degree of expertise to effectively establish reliable experiments and analyze data. Differential scanning fluorimetry (DSF) is being increasingly used as a robust method for initial screening of proteins for interacting small molecules, either for identifying physiological partners or for hit discovery. This technique has the advantage that it requires only a PCR machine suitable for quantitative PCR, and so suitable instrumentation is available in most institutions; an excellent range of protocols are already available; and there are strong precedents in the literature for multiple uses of the method. Past work has proposed several means of calculating dissociation constants from DSF data, but these are mathematically demanding. Here, we demonstrate a method for estimating dissociation constants from a moderate amount of DSF experimental data. These data can typically be collected and analyzed within a single day. We demonstrate how different models can be used to fit data collected from simple binding events, and where cooperative binding or independent binding sites are present. Finally, we present an example of data analysis in a case where standard models do not apply. These methods are illustrated with data collected on commercially available control proteins, and two proteins from our research program. Overall, our method provides a straightforward way for researchers to rapidly gain further insight into protein-ligand interactions using DSF.

Differential scanning fluorimetry is a widely used method for screening libraries of small molecules for interactions with proteins. Here, we present a straightforward method to extend these analyses to provide an estimate of the dissociation constant between a small molecule and its protein partner.

蛋白质 - 配体相互作用采用差示扫描荧​​光法测定

A wide range of methods are currently available for determining the dissociation constant between a protein and interacting small molecules. However, most ...

微量法测定蛋白浓度使用NanoDrop 2000C分光光度计

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