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。

<table><tbody><tr><td>NanoDrop 2000c Spectrophotometer</td><td>Thermo Fisher Scientific, Inc.</td><td></td><td></td></tr><tr><td>Laboratory wipes</td><td></td><td></td><td></td></tr><tr><td>Pierce BCA Protein Assay Kit</td><td>Thermo Fisher Scientific, Inc.</td><td>23250</td><td>Reducing agent compatible</td></tr><tr><td>PR-1 Reconditioning Kit</td><td>Thermo Fisher Scientific, Inc.</td><td></td><td></td></tr></tbody></table>

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

Counting Proteins in Single Cells with Addressable Droplet Microarrays

Often cellular behavior and cellular responses are analyzed at the population level where the responses of many cells are pooled together as an average result masking the rich single cell behavior within a complex population. Single cell protein detection and quantification technologies have made a remarkable impact in recent years. Here we describe a practical and flexible single cell analysis platform based on addressable droplet microarrays. This study describes how the absolute copy numbers of target proteins may be measured with single cell resolution. The tumor suppressor p53 is the most commonly mutated gene in human cancer, with more than 50% of total cancer cases exhibiting a non-healthy p53 expression pattern. The protocol describes steps to create 10 nL droplets within which single human cancer cells are isolated and the copy number of p53 protein is measured with single molecule resolution to precisely determine the variability in expression. The method may be applied to any cell type including primary material to determine the absolute copy number of any target proteins of interest.

Here we present addressable droplet microarrays (ADMs), a droplet array based method able to determine absolute protein abundance in single cells. We demonstrate the capability of ADMs to characterize the heterogeneity in expression of the tumor suppressor protein p53 in a human cancer cell line.

用可寻址滴微阵列计算单细胞中的蛋白质

Often cellular behavior and cellular responses are analyzed at the population level where the responses of many cells are pooled together as an average ...

科研

癌症研究

Standards for Quantitative Metalloproteomic Analysis Using Size Exclusion ICP-MS

Metals are essential for protein function as cofactors to catalyze chemical reactions. Disruption of metal homeostasis is implicated in a number of diseases including Alzheimer's and Parkinson's disease, but the exact role these metals play is yet to be fully elucidated. Identification of metalloproteins encounters many challenges and difficulties. Here we report an approach that allows metalloproteins in complex samples to be quantified. This is achieved using size exclusion chromatography coupled with inductively coupled plasma - mass spectrometry (SEC-ICP-MS). Using six known metalloproteins, the size exclusion column can be calibrated and the respective trace elements (iron, copper, zinc, cobalt, iodine) can be used for quantification. SEC-ICP-MS traces of human brain and plasma are presented. The use of these metalloprotein standards provides the means to quantitatively compare metalloprotein abundances between biological samples. This technique is poised to help shed light on the role of metalloproteins in neurodegenerative disease as well as other diseases where imbalances in trace elements are implicated.

Size exclusion chromatography hyphenated with inductively coupled plasma - mass spectrometry (ICP-MS) is a powerful tool to measure changes in the abundance of metalloproteins directly from biological samples. Here we describe a set of metalloprotein standards used to estimate molecular mass and the amount of metal associated with unknown proteins.

用尺寸排阻ICP-MS标准定量分析Metalloproteomic

Metals are essential for protein function as cofactors to catalyze chemical reactions. Disruption of metal homeostasis is implicated in a number of ...

化学

Evaluation of Polymeric Gene Delivery Nanoparticles by Nanoparticle Tracking Analysis and High-throughput Flow Cytometry

Non-viral gene delivery using polymeric nanoparticles has emerged as an attractive approach for gene therapy to treat genetic diseases1 and as a technology for regenerative medicine2. Unlike viruses, which have significant safety issues, polymeric nanoparticles can be designed to be non-toxic, non-immunogenic, non-mutagenic, easier to synthesize, chemically versatile, capable of carrying larger nucleic acid cargo and biodegradable and/or environmentally responsive. Cationic polymers self-assemble with negatively charged DNA via electrostatic interaction to form complexes on the order of 100 nm that are commonly termed polymeric nanoparticles. Examples of biomaterials used to form nanoscale polycationic gene delivery nanoparticles include polylysine, polyphosphoesters, poly(amidoamines)s and polyethylenimine (PEI), which is a non-degradable off-the-shelf cationic polymer commonly used for nucleic acid delivery1,3 . Poly(beta-amino ester)s (PBAEs) are a newer class of cationic polymers4 that are hydrolytically degradable5,6 and have been shown to be effective at gene delivery to hard-to-transfect cell types such as human retinal endothelial cells (HRECs)7, mouse mammary epithelial cells8, human brain cancer cells9 and macrovascular (human umbilical vein, HUVECs) endothelial cells10.
A new protocol to characterize polymeric nanoparticles utilizing nanoparticle tracking analysis (NTA) is described. In this approach, both the particle size distribution and the distribution of the number of plasmids per particle are obtained11. In addition, a high-throughput 96-well plate transfection assay for rapid screening of the transfection efficacy of polymeric nanoparticles is presented. In this protocol, poly(beta-amino ester)s (PBAEs) are used as model polymers and human retinal endothelial cells (HRECs) are used as model human cells. This protocol can be easily adapted to evaluate any polymeric nanoparticle and any cell type of interest in a multi-well plate format.

A protocol for nanoparticle tracking analysis (NTA) and high-throughput flow cytometry to evaluate polymeric gene delivery nanoparticles is described. NTA is utilized to characterize the nanoparticle particle size distribution and the plasmid per particle distribution. High-throughput flow cytometry enables quantitative transfection efficacy evaluation for a library of gene delivery biomaterials.

高分子基因传递纳米粒子的纳米粒子跟踪分析和高通量流式细胞仪评价

Non-viral gene delivery using polymeric nanoparticles has emerged as an attractive approach for gene therapy to treat genetic diseases1 and as a ...

生物工程

UV-Vis Spectroscopic Characterization of Nanomaterials in Aqueous Media

The physicochemical characterization of nanomaterials (NMs) is often an analytical challenge, due to their small size (at least one dimension in the nanoscale, i.e. 1&#8211;100 nm), dynamic nature, and diverse properties. At the same time, reliable and repeatable characterization is paramount to ensure safety and quality in the manufacturing of NM-bearing products. There are several methods available to monitor and achieve reliable measurement of nanoscale-related properties, one example of which is Ultraviolet-Visible Spectroscopy (UV-Vis). This is a well-established, simple, and inexpensive technique that provides non-invasive and fast real-time screening evaluation of NM size, concentration, and aggregation state. Such features make UV-Vis an ideal methodology to assess the proficiency testing schemes (PTS) of a validated standard operating procedure (SOP) intended to evaluate the performance and reproducibility of a characterization method. In this paper, the PTS of six partner laboratories from the H2020 project ACEnano were assessed through an interlaboratory comparison (ILC). Standard gold (Au) colloid suspensions of different sizes (ranging 5&#8211;100 nm) were characterized by UV-Vis at the different institutions to develop an implementable and robust protocol for NM size characterization.

This study presents the benchmarking results for an interlaboratory comparison (ILC) designed to test the standard operating procedure (SOP) developed for gold (Au) colloid dispersions characterized by ultraviolet-visible Spectroscopy (UV-Vis), amongst six partners from the H2020 ACEnano project for sample preparation, measurement, and analysis of the results.

水介质中纳米材料的紫外-可见光谱表征

The physicochemical characterization of nanomaterials (NMs) is often an analytical challenge, due to their small size (at least one dimension in the ...

工程学

使用微流体扩展质量亮度分析浓度范围

Analysis of Protein Complex Formation at Micromolar Concentrations by Coupling Microfluidics with Mass Photometry

Mass photometry is a versatile mass measurement technology that enables the study of biomolecular interactions and complex formation in solution without labels. Mass photometry is generally suited to analyzing samples in the 100 pM-100 nM concentration range. However, in many biological systems, it is necessary to measure more concentrated samples to study low-affinity or transient interactions. Here, we demonstrate a method that effectively expands the range of sample concentrations that can be analyzed by mass photometry from nanomolar to tens of micromolar. 
In this protocol, mass photometry is combined with a novel microfluidics system to investigate the formation of protein complexes in solution in the micromolar concentration range. With the microfluidics system, users can maintain a sample at a desired higher concentration followed by dilution to the nanomolar range - several milliseconds prior to the mass photometry measurement. Due to the speed of the dilution, data is obtained before the equilibrium of the sample has shifted (i.e., dissociation of the complex). 
The technique is applied to measure interactions between an immunoglobulin G (IgG) antibody and the neonatal Fc receptor, showing the formation of high-order complexes that were not quantifiable with static mass photometry measurements. 
In conclusion, the combination of mass photometry and microfluidics makes it possible to characterize samples in the micromolar concentration range and is proficient in measuring biomolecular interactions with weaker affinities. These capabilities can be applied in a range of contexts - including the development and design of biotherapeutics - enabling thorough characterization of diverse protein-protein interactions.

作者聚焦：使用 MassFluidix 技术表征溶液中低亲和力蛋白质相互作用

This protocol combines mass photometry with a novel microfluidics system to investigate low-affinity protein-protein interactions. This approach is based on the rapid dilution of highly concentrated complexes in solution, which enables low-affinity measurements and broadens the applicability of mass photometry.

通过将微流体与质量光度法相结合来分析微摩尔浓度下的蛋白质复合物形成

Mass photometry is a versatile mass measurement technology that enables the study of biomolecular interactions and complex formation in solution without ...

生物化学

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 ...

生物学

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 ...

Methods to Discover Alternative Promoter Usage and Transcriptional Regulation of Murine Bcrp1

Gene expression in different tissues is often controlled by alternative promoters that result in the synthesis of mRNA with unique &#8212; usually untranslated &#8212; first exons. Bcrp1 (Abcg2), the murine orthologue of the ABC transporter Breast Cancer Resistance Protein (BCRP, ABCG2), has at least four alternative promoters that are designated by the corresponding four alternative first exons produced: E1U, E1A, E1B, and E1C. Herein, in-silico protocols are presented to predict alternative promoter usage for Bcrp1. Furthermore, reporter assay methods are described to produce reporter constructs for alternative promoters and to determine the functionality of putative promoters upstream of the alternative first exons that are identified.

With the murine ABC transporter Bcrp1 (Abcg2) as an example, in-silico protocols are presented to detect alternative promoter usage in genes expressed in mouse tissues, and to evaluate the functionality of the alternative promoters identified using reporter assays.

微量法测定蛋白浓度使用NanoDrop 2000C分光光度计

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水介质中纳米材料的紫外-可见光谱表征

通过将微流体与质量光度法相结合来分析微摩尔浓度下的蛋白质复合物形成

通过将微流体与质量光度法相结合来分析微摩尔浓度下的蛋白质复合物形成

通过将微流体与质量光度法相结合来分析微摩尔浓度下的蛋白质复合物形成

NanoDrop微量核酸的定量

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

方法发现小鼠BCRP1的另类用法子和转录调控