Name: nonradioactive assay to measure polynucleotide phosphorylation of small nucleotide substrates
Uploaded: 2020-05-08T15:00:00
Description: Watch this Scientific Journal Video about Nonradioactive Assay to Measure Polynucleotide Phosphorylation of Small Nucleotide Substrates at JoVE.com

我们感谢安德鲁·西克马博士和安德里亚·卡明斯基对这份手稿的批判性解读。这项工作得到了美国国家卫生研究院校内研究项目的支持;美国国家环境卫生科学研究所（NIEHS;ZIA ES103247 至 R.E.S） 和加拿大卫生研究所 （CIHR; 146626 到 M.C.P）。

1. 准备
<ol><li>准备缓冲液和试剂。<ol>
<li>通过组合 20 μL 的 1 M Tris （pH = 8.0）、40 μL 的 5 M 氯化钠、2.5 μL 的 2 M 氯化镁、100 μL 的 50%（v/v）甘油和无 1 mL 的 RNase 水，制成 1x 反应缓冲液，达到 1 mL 的总体积。</li>
		<li>通过组合 4.8 g 尿素、200 μL 的 1 M Tris （pH = 8.0）、20 μL 的 0.5 M EDTA （pH = 8.0）、0.5 mL 的 1% （w/v） 肉酚蓝色和无 Rse-0 水，使尿素加载染料达到 10 mL 的总体积。</li>
		<li>通过组合 108 g ...

<ol>
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所述是测量Grc3 PNK在荧光标记核苷酸基质基质上激酶活性的测定方法。该协议可以通过调整反应缓冲液和寡核苷酸基质来应用于其他PNK酶的特性。例如，协议要求跟踪量为 EDTA。添加EDTA是有益的，原因有二：首先，这种方法有利于镁结合Grc3，防止酶与混合物中微量的污染金属结合。其次，少量的EDTA抑制污染金属依赖性核糖核酸的活动，而不会破坏相关的Las1金属独立核糖核糖的活性。EDTA 的浓度可...

多核苷酸激酶（PNK）在许多DNA和RNA处理途径中起着至关重要的作用，如DNA修复和核糖体组装1、2、3、4、5。,2,3,4,5这些基本酶催化终端（伽马）单磷酸盐从核苷三磷酸盐（NTP，最常见的ATP）转移到核苷酸基质的5'羟基端。PNK最具有最良好特征之一是细菌T4,PNK，它具有广泛的基质特异性，被分子生物学实验室大量利用，将放射性同位素标签整合到DNA或RNA基质,,,6、7、8、9、10、11、127,8的5个术语中。91011PNK酶的另一个例子是CLP1，它存在于尤卡里亚、尤菌和阿奇亚，并牵连到几个RNA处理途径4，13，14，15。,14,154,
从历史上看，大多数测量多核苷酸激酶活性的测定都依赖于放射性同位素标记和随后的自体造影术,5，16。近年来，还开发了一些用于测量PNK活性的附加检测方法，包括单分子方法、微芯片电泳、分子信标，以及基于,色度和发光的测定17、18、19、20、21、22。,18,19,2021,22虽然许多新方法提供了增强的检测限值并避免使用放射性，但每种方法都有缺点，例如成本、对固定树脂的依赖以及基板选择的限制。
Grc3是一种多核苷酸激酶，在核糖体前RNA2、3、23,3,的处理中起着关键作用。Grc3与内白蛋白酶Las1形成一个义务复合体，它切割了前核糖体RNA3的内部转录空间2（ITS2）。Las1的 ITS2 的裂解产生一种含有 5' 羟基的产品，随后由 Grc3 激酶3 磷酸化。为了研究Grc3的核苷酸和基质特异性，需要一种廉价的测定方法，允许对不同的寡核苷酸基质进行检测。因此，利用荧光标记基材开发了PNK磷酸化测定。该测定成功地用于确定Grc3可以利用任何NTP进行磷酸转移活性，但有利于ATP24。该协议调整原始测定，以测量Grc3的PNK活性，其RNA模拟其前核糖体RNA基质（SC-ITS2，表1）。这种基于荧光的方法的一个具有挑战性的方面是依靠大型聚丙烯酰胺凝胶来有效解决磷酸化和非磷化基材。该协议提供了有关如何倒这些大凝胶的具体细节，并避免在这样做时常见的陷阱。
使用RNA需要特别小心，因为它极易降解。有简单的预防措施，可以采取，以限制核糖核酸酶污染。单独的RNA工作站，可以很容易地用含有RNase抑制剂的清洁剂处理，通常是有帮助的。处理样品时始终佩戴手套，并且必须使用无 RNase 认证的耗材。由于水是另一种常见的污染源，因此最好使用新鲜纯净的水，并使用 0.22 μm 过滤器对所有溶液进行消毒。

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	<tbody>
		<tr>
			<td>0.4 mm 34-well comb</td>
			<td>BioRad</td>
			<td>1653848</td>
			<td></td>
		</tr>
		<tr>
			<td>0.4 mm spacer</td>
			<td>BioRad</td>
			<td>1653812</td>
			<td></td>
		</tr>
		<tr>
			<td>0.5 M EDTA ph 8.0</td>
			<td>KD Medical</td>
			<td>RGF-3130</td>
			<td></td>
		</tr>
		<tr>
			<td>1M Magnesium Chloride</td>
			<td>KD Medical</td>
			<td>CAC-5290</td>
			<td></td>
		</tr>
		<tr>
			<td>1M Tris pH 8.0</td>
			<td>KD Medical</td>
			<td>RGF-3360</td>
			<td></td>
		</tr>
		<tr>
			<td>40% Acrylamide/Bis Solution 29:1</td>
			<td>BioRad</td>
			<td>1610146</td>
			<td></td>
		</tr>
		<tr>
			<td>5M Sodium Chloride</td>
			<td>KD Medical</td>
			<td>RGF-3720</td>
			<td></td>
		</tr>
		<tr>
			<td>ammonium persulfate (APS)</td>
			<td>BioRad</td>
			<td>161-0700</td>
			<td></td>
		</tr>
		<tr>
			<td>ATP</td>
			<td>Sigma</td>
			<td>A2383-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>boric acid</td>
			<td>Sigma</td>
			<td>B0394</td>
			<td></td>
		</tr>
		<tr>
			<td>bromophenol blue sodium salt</td>
			<td>Sigma</td>
			<td>B5525-5G</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Plates</td>
			<td>Thomas Scientific</td>
			<td>1188K51</td>
			<td></td>
		</tr>
		<tr>
			<td>Hoefer SQ3 Sequencer</td>
			<td>Hoefer</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Image J Software</td>
			<td>N/A</td>
			<td>N/A</td>
			<td><a href="https://imagej.nih.gov/ij/" target="_blank">https://imagej.nih.gov/ij/</a></td>
		</tr>
		<tr>
			<td>Labeled RNA oligonucleotides</td>
			<td>IDT</td>
			<td>Custom Order</td>
			<td></td>
		</tr>
		<tr>
			<td>Pharmacia EPS 3500 Power Supply</td>
			<td>Pharmacia</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Steriflip 0. 22 um Filter</td>
			<td>Millipore</td>
			<td>5FCP00525</td>
			<td></td>
		</tr>
		<tr>
			<td>TEMED</td>
			<td>BioRad</td>
			<td>161-0800</td>
			<td></td>
		</tr>
		<tr>
			<td>tris base</td>
			<td>Sigma</td>
			<td>TRIS-RO</td>
			<td></td>
		</tr>
		<tr>
			<td>Typhoon FLA 9500 gel imager</td>
			<td>GE Healthcare</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultra Pure DEPC Water</td>
			<td>Invitrogen</td>
			<td>750023</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultra Pure Glycerol</td>
			<td>Invitrogen</td>
			<td>19E1056865</td>
			<td></td>
		</tr>
		<tr>
			<td>urea</td>
			<td>Fisher Chemical</td>
			<td>U15-500</td>
			<td></td>
		</tr>
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</table>

nonradioactive assay to measure polynucleotide phosphorylation of small nucleotide substrates

多核苷酸激酶（PNKs）是催化DNA和RNA寡核苷酸5'羟基端磷酸化的酶。可以直接或间接地对PNK的活动进行量化。此处介绍的是一种直接的体外测量 PNK 活性的方法，它依赖于荧光标记的寡核苷酸基质和聚丙烯酰胺凝胶电泳。这种方法提供磷酸化产品的分辨率，同时避免使用放射性标记基板。协议详细介绍了如何设置磷酸化反应，准备和运行大型聚丙烯酰胺凝胶，并量化反应产品。这种测定技术上最具挑战性的部分是浇注和运行大型聚丙烯酰胺凝胶;因此，提供了克服共同困难的重要细节。该协议针对Grc3进行了优化，Grk是一种PNK，它结合其结合伙伴Las1核酸酶组装成一个义务性前核糖体RNA处理复合物。然而，此协议可以适应测量其他PNK酶的活性。此外，这种测定也可以被修改，以确定反应的不同成分的影响，如核苷三磷酸盐，金属离子和寡核苷酸。

图1中显示了ATP滴定与固定数量的Las1-Grc3复合物的成功代表性 变性凝胶。酶的加入导致SC-ITS2 RNA基质的Las1中导RNA裂解，导致定义的RNA片段（5-OH C2 RNA）。添加ATP后，C2 RNA片段由Grc3 PNK（5-P C2 RNA）磷酸化。在变性凝胶中，磷酸化RNA的迁移速度比其未磷化RNA的迁移速度要快。如图 2所示，C2RNA片段的磷酸化可以通过绘制未磷化和磷酸?...

Watch this Scientific Journal Video about Nonradioactive Assay to Measure Polynucleotide Phosphorylation of Small Nucleotide Substrates at JoVE.com

Nonradioactive Assay to Measure Polynucleotide Phosphorylation of Small Nucleotide Substrates

该协议描述了一种非放射性检测，用于测量小DNA和RNA基质上多核苷酸激酶（PNKs）的激酶活性。

测量小核苷酸基质的多核苷酸磷酸化的非放射性测定

Polynucleotide kinases (PNKs) are enzymes that catalyze the phosphorylation of the 5&#39; hydroxyl end of DNA and RNA oligonucleotides. The activity of ...

该方法可以通过一种特殊的多核苷酸激酶来回答有关DNA或RNA分子五主要端磷酸化的关键问题。该技术的主要优点是具有检测DNA和RNA基质中非常小变化的分辨率。首先准备RNA酶激酶反应。对于每次反应，结合一个微升的500纳米摩尔RNA基板。8.3 微升 130 纳米摩尔最后一个， GRC 三和 0.2 微升 5 毫摩尔 EDTA.将热块设置为 37 摄氏度。然后将浓度序列中的 ATP 子库存的 0.5 微升与一个 RNA 酶混合物混合，将反应放在热块上。继续混合反应，并每隔十秒钟将其放置在热块上。在热块上孵育60分钟后，用10微升尿素装载染料将每次反应都淬火。立即执行下游分析，或将反应储存在负 20 摄氏度，以在以后进行分析。要准备 15% 变性丙烯酰胺凝胶溶液，请将 22.5 毫升预混合 40%29 与一个丙烯酰胺/二丙烯酰胺溶液混合。六毫升的十个 x TBE 。28.8克尿素。和RNase无水总体积为59毫升。然后轻轻搅拌溶液。在微波炉中加热溶液20秒。搅拌它，并立即返回到微波炉再20秒。轻轻搅拌溶液，直到尿素完全溶解。将玻璃烧杯放入浅水浴中，含冷水五分钟，确保玻璃烧杯周围的冷水水平高于玻璃烧杯内溶液的水平。当溶液冷却时，使用0.2 2微米一次性过滤装置过滤并去气，以去除颗粒物和微气泡。用肥皂和水清洗短而长的玻璃板，然后用95%乙醇喷洒每个盘子，擦拭玻璃以去除任何水分。将长板放在盒子顶部，将其从工作台顶部提升。然后沿板的长边放置 0.4 毫米的分位器。将短板放在长板的顶部，确保短板、长板和垫片的边缘对齐。然后用三个均匀的金属夹夹夹住每一侧。将 24 微升 TEMED 添加到丙烯酰胺溶液中并混合。然后加入 600 微升 10% APS，立即将溶液倒在玻璃板之间。在玻璃板三明治之间浇灌丙烯酰胺溶液可能非常具有挑战性。为避免气泡，在浇注溶液时点按玻璃板三明治。小心地在玻璃板三明治的顶部添加一个干净的 32 井梳子。使丙烯酰胺聚合30分钟。要运行凝胶，请将热块设置为 75 摄氏度。拆下金属夹。彻底清洗和干燥玻璃板三明治。将板夹层和凝胶装置放置，使短板朝前，通过将 100 毫升 10 X TBE 与 1.9 升 RNase 无水相结合，准备 0.5 X TBE 运行缓冲液。将 600 毫升的运行缓冲液添加到仪器的上部和下腔。轻轻地从凝胶上取下梳子，然后用注射器彻底冲洗孔。以 50 瓦的功率预运行凝胶 30 分钟。然后再次冲洗井。后旋转淬火反应，并在75摄氏度孵育三分钟。重复脉冲旋转，立即将每个样品的 10 微升加载到凝胶上。然后在50瓦下运行凝胶三小时。运行完成后，关闭电源并排空设备上腔。清洗和干燥玻璃板三明治的外侧。然后用铝箔盖住它，然后转移到激光扫描仪进行成像。将玻璃板三明治安装到激光扫描仪的舞台上。为凝胶设置所需地板的激发和发射波长并成像凝胶。此处显示的是 ATP 滴定中具有代表性的成功变性凝胶，其固定量为最后一个 GRC 三复合体。酶的加入导致最后一个中化RNA裂解的糖精化SAC2两个RNA基质，导致一个定义的RNA片段。加入ATP后，C2 RNA片段由GrC3 PNK磷酸化。为了可视化C2 RNA片段的磷酸化，针对ATP浓度绘制了未磷酸化和磷酸化C2RNA的相对量。我们代表不成功的变性凝胶显示在这里。21核苷酸RNA基质含有降解产物，与磷酸化产物重叠，无法准确量化磷酸化。相比之下，最短的RNA降解产物可以成功分析，因为凝胶的这一区域不包含任何额外的RNA物种，这妨碍了其磷酸化对应物的精确定量。尝试此协议时，要记住的最重要的事情是冲洗凝胶的孔，以确保样品的均匀加载。按照这个程序，可以进行RNA周转实验。为了测量RNA降解率，磷酸化通常标志着RNA降解的开始。该技术为回答有关多核苷酸激酶的活动特异性和动力学的详细问题铺平了道路。

Research

JoVE Journal

Biochemistry

Oligopeptide Competition Assay for Phosphorylation Site Determination

寡肽竞争测定磷酸化位点测定

Protein phosphorylation at specific sites determines its conformation and interaction with other molecules. Thus, protein phosphorylation affects ...

科研

生物化学

Quantitative Immunofluorescence to Measure Global Localized Translation

定量荧光测量全球本地化翻译

The mechanisms regulating mRNA translation are involved in various biological processes, such as germ line development, cell differentiation, and ...

Assay for Phosphorylation and Microtubule Binding Along with Localization of Tau Protein in Colorectal Cancer Cells

大肠癌细胞中磷酸化和微管结合与 Tau 蛋白定位的测定

The microtubule-associated protein tau is a neuronal protein that localizes mostly in axons. Generally tau is essential for normal neuronal functioning ...

Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay

Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay

用体外激酶法鉴定细胞周期蛋白依赖性激酶1特异磷酸化部位

Cyclin-dependent kinase 1 (Cdk1) is a master controller for the cell cycle in all eukaryotes and phosphorylates an estimated 8 - 13% of the proteome; ...

Proofreading and DNA Repair Assay Using Single Nucleotide Extension and MALDI-TOF Mass Spectrometry Analysis

单核苷酸延长和 MALDI 质谱分析校对及 DNA 修复方法

The maintenance of the genome and its faithful replication is paramount for conserving genetic information. To assess high fidelity replication, we have ...

Dual DNA Rulers to Study the Mechanism of Ribosome Translocation with Single-Nucleotide Resolution

双DNA标尺研究单核苷酸分辨率的核糖体易位机制

The ribosome translocation refers to the ribosomal movement on the mRNA by exactly three nucleotides (nt), which is the central step in protein synthesis. ...

Use of Microscale Thermophoresis to Measure Protein-Lipid Interactions

使用微尺度热泳测量蛋白质-脂质相互作用

The ability to determine the binding affinity of lipids to proteins is an essential part of understanding protein-lipid interactions in membrane ...

Using an Extracellular Flux Analyzer to Measure Changes in Glycolysis and Oxidative Phosphorylation during Mouse Sperm Capacitation

使用细胞外通量分析仪测量小鼠精子发作期间糖解和氧化磷酸化的变化

Mammalian sperm acquire fertilization capacity in the female reproductive tract in a process known as capacitation. Capacitation-associated processes ...

Measuring Nucleotide Binding to Intact, Functional Membrane Proteins in Real Time

实时测量核苷酸与完整功能性膜蛋白的结合

We have developed a method to measure binding of adenine nucleotides to intact, functional transmembrane receptors in a cellular or membrane environment. ...

A High-Throughput Enzyme-Coupled Activity Assay to Probe Small Molecule Interaction with the dNTPase SAMHD1

Sterile alpha motif and HD domain-containing protein 1 (SAMHD1) is a pivotal regulator of intracellular deoxynucleoside triphosphate (dNTP) pools, as this ...