Name: a "dual-addition" calcium fluorescence assay for the high-throughput screening of recombinant g protein-coupled receptors
Uploaded: 2022-12-02T13:20:00
Description: Watch this Scientific Journal Video about A "Dual-Addition" Calcium Fluorescence Assay for the High-Throughput Screening of Recombinant G Protein-Coupled Receptors at JoVE.com

这项工作得到了美国农业部-NIFA-AFRI动物健康和福祉奖（奖项编号2022-67015-36336，PVP [项目总监]）的支持，以及德克萨斯州A&M AgriLife研究昆虫媒介疾病资助计划（FY'22-23）的竞争性资金给P.V.P. TAMU农业与生命科学学院的A.W.E.S.O.M.E.教师小组因帮助编辑手稿而受到认可。 补充表S2 包含来自德克萨斯A&M大学James Sacchettini博士实验室和德克萨斯A&M AgriLife Research的内部随机小分子库的数据。

1. 电池维护
注意：一种稳定表达来自 R. MICROPLUS的激肽受体的CHO-K1细胞系，名为BMLK3，由Holmes等人开发16。细胞系开发的细节详见其他专题14。以下所有步骤均在无菌条件下在II类生物安全柜中进行。
<ol><li>在选择性培养基（含有 10% 胎牛血清 [FBS] 和 800 μg/mL G418 硫酸盐的 F-12K 培养基）中培养重组细胞系，以确保?...

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HTS的目标是通过筛选大量小分子来识别命中分子。因此，本例的结果仅代表常规HTS实验的一小部分。此外，鉴定出的命中分子需要在下游检测中验证，例如在同一重组细胞系和仅携带空载体的 CHO-K1 细胞系上进行剂量依赖性检测，可以同时进行以保存小分子。细胞毒性测定将有助于证明第二次添加时缺乏反应不是由于细胞死亡率。应进行其他生物测定以验证 体外分离组织中的活性，或者通?...

G蛋白偶联受体（GPCRs）存在于酵母到人类，是许多生物体中最大的受体超家族1。它们在调节动物几乎所有生物过程中起着关键作用。节肢动物的基因组中有50-200个GPCR，这意味着它们代表了最大的膜受体超家族2。它们根据序列相似性和功能分为六大类，A-F3。GPCR转导各种细胞外信号，例如激素，神经肽，生物胺，谷氨酸，质子，脂糖蛋白和光子的信号4。GPCR与异源三聚体G蛋白（Gα，Gβ和Gγ）偶联以传递下游信号。与Gαs 或Gαi / o 蛋白偶联的GPCR通过激活或抑制腺苷环化酶分别增加或降低细胞内3'，5'-环磷酸腺苷（cAMP）水平。与Gαq/11 偶联的GPCR通过激活磷脂酶C（PLC）-肌醇-1，4，5-三磷酸（IP3）途径，诱导钙从内质网钙储存中释放。与Gα12/13 偶联的GPCR激活RhoGTP酶核苷酸交换因子5，6。GPCR是超过50%的人类药物和杀螨剂amitraz4的靶标。由于GPCR转导了如此多样化的信号，它们是开发破坏无脊椎动物特异性生理功能的新型杀虫剂的有希望的目标。
HTS的目标是识别可以调节受体功能的命中分子。HTS 涉及检测开发、小型化和自动化7。节肢动物神经肽GPCR参与大多数生理功能，如发育、蜕皮和蜕皮、排泄、能量动员和繁殖4。节肢动物和后生动物的大多数神经肽GPCR通过钙信号级联反应2，6，8，9，10发出信号，例如在黑腿蜱肩突硬蜱的肌抑制肽和SIFamide受体中;它们的配体在后肠运动测定中具有拮抗作用，SIF引起收缩，MIP抑制收缩11，12。黄热病蚊子的NPY样受体埃及伊蚊调节雌性宿主寻找13。与其他替代钙动员测定（例如水杉钙生物发光测定14）相比，钙荧光测定易于执行，不需要转染其他重组钙检测蛋白，并且具有成本效益。与在水杉钙生物发光测定中获得的快速动力学信号相比，钙荧光测定产生更长的信号14，15.
在这里的示例中，来自牛瘟蜱的激 肽受体Rhipicephalus microplus在CHO-K1细胞系中重组表达并用于钙荧光测定。在 R. microplus中仅发现一个激肽受体基因;受体通过Gq蛋白依赖性信号通路发出信号，并触发Ca2+ 从钙储存到细胞内空间的外排16。该过程可以通过荧光团进行检测和定量，荧光团在结合钙离子时引发荧光信号（图1）。
激肽受体是无脊椎动物特异性GPCR，属于A类视紫红质样受体。激肽是一种古老的信号神经肽，存在于软体动物、甲壳纲、昆虫和螨科4，17，18 中。鞘翅目动物（甲虫）缺乏激肽信号系统;在蚊子埃及伊蚊中，只有一个激肽受体结合三个伊蚊素，而黑腹果蝇有一个激肽受体，果蓿素作为独特的配体19，20，21。脊椎动物中没有同源激肽或激肽受体。虽然激肽的确切功能在蜱中尚不清楚，但激肽受体RNAi沉默的雌性R. microplus显示出显着降低的生殖适应性22。激肽是昆虫中的多效性肽。在黑腹果蝇中，它们参与中枢和周围神经系统调节系统23，蜕皮前期24，喂养25，代谢26和睡眠活动模式26，27，以及幼虫运动28。激肽调节蚊子埃及伊蚊的后肠收缩、利尿和摄食 29，30，31。激肽具有保守的C端五肽Phe-X1-X2-Trp-Gly-NH2，这是生物活性所需的最小序列32。节肢动物的特异性、内源性配体的小尺寸（使其易于小分子干扰）以及昆虫中的多效性功能使激肽受体成为害虫防治的有希望的靶标4。
“双重加成”测定（图2）允许在同一HTS测定15中鉴定激动剂或拮抗剂。它改编自制药行业中通常用于药物发现的“双重添加”测定33。简而言之，与溶剂对照的应用相比，首次将药物添加到细胞板中允许在检测到更高的荧光信号时鉴定化学库中的潜在激动剂。用这些小分子孵育5分钟后，将已知的激动剂（激肽）施用于所有孔。与在第一次添加中接受溶剂的对照孔相比，那些从药物板随机接受拮抗剂的孔在加入激动剂时显示出较低的荧光信号。然后，该测定允许鉴定具有相同细胞的潜在激动剂和拮抗剂。在标准的HTS项目中，这些命中分子将通过剂量反应测定和额外的生物活性测定进一步验证，此处未显示。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64505/64505fig01.jpg"> 图1：钙荧光测定机理图示。Gq蛋白触发细胞内钙信号通路。激肽受体（G蛋白偶联受体）在CHO-K1细胞中重组表达。当激动剂配体与受体结合时，与激肽受体相关的Gq蛋白激活PLC，从而催化PIP2分子转化为IP3和DAG。然后，IP 3 与内质网表面的 IP3 R 结合，导致 Ca 2+ 释放到细胞质中，其中Ca 2+ 离子与荧光团结合并引发荧光信号。荧光信号可以通过在490nm处激发并在514nm处检测获得。缩写：GPCR = G蛋白偶联受体;PLC = 磷脂酶 C;PIP2 = 磷脂酰肌醇 4，5-二磷酸;IP3 = 肌醇三磷酸;DAG = 二酰基甘油;IP3 R = IP3 受体。用 BioRender.com 创建。<a href="https://www.jove.com/files/ftp_upload/64505/64505fig01large.jpg" target="_blank">请点击此处查看此图的大图。</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64505/64505fig02.jpg"> 图 2：在 CHO-K1 细胞中表达的 G 蛋白偶联受体上高通量筛选小分子的工作流程。 （A）使用液体处理系统（25μL/孔）将稳定表达激肽受体的重组CHO-K1细胞添加到384孔板（10，000个细胞/孔）中，并在加湿的CO2培养箱中孵育12-16小时。）使用液体处理系统将含有荧光染料（25μL/孔）的测定缓冲液加入细胞板中。将板在37°C下孵育30分钟30分钟，并在室温下再平衡30分钟。 （C）用读板器测量每个孔中细胞的背景荧光信号。（D） 使用液体处理系统将来自 384 孔文库板和空白溶剂（均为 0.5 μL/孔）的药物溶液添加到细胞测定板中。（E）加入药物溶液后立即用酶标仪测量细胞钙荧光反应;引起高于平均水平荧光信号的化合物被挑选出来作为激动剂命中。在步骤G期间加入肽激动剂后，揭示了阻断GPCR的拮抗剂命中（下图）。（F）在同一测定板中，将细胞与筛选化合物孵育5分钟后，将蜱激肽受体的内源性激动剂肽Rhimi-K-1（QFSPWGamide）加入到每个孔（1μM）中。（G）加入激动剂肽后立即用酶标仪测量细胞荧光反应。选择抑制荧光信号的化合物作为拮抗剂命中。缩写：GPCR = G蛋白偶联受体;RT = 室温;RFU = 相对荧光单位。用 BioRender.com 创建。<a href="https://www.jove.com/files/ftp_upload/64505/64505fig02large.jpg" target="_blank">请点击此处查看此图的大图。</a>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.25% trypsin-EDTA</td>
			<td>Gibco Invitrogen</td>
			<td>15050-065</td>
			<td>with phenol red</td>
		</tr>
		<tr>
			<td>0.4% trypan blue</td>
			<td>MilliporeSigma</td>
			<td>T8154</td>
			<td>liquid, sterile</td>
		</tr>
		<tr>
			<td>1.5 mL microcentrifuge tubes</td>
			<td>Thermo Fisher</td>
			<td>AM12400</td>
			<td>RNase-free Microfuge Tubes</td>
		</tr>
		<tr>
			<td>5 mL serological pipette</td>
			<td>Corning</td>
			<td>29443-045</td>
			<td>Corning Costar Stripette individually wrapped&#160;</td>
		</tr>
		<tr>
			<td>10 mL serological pipette</td>
			<td>Corning</td>
			<td>29443-047</td>
			<td>Corning Costar Stripette individually wrapped&#160;</td>
		</tr>
		<tr>
			<td>15 mL conical tubes</td>
			<td>Falcon</td>
			<td>352196</td>
			<td>sterile</td>
		</tr>
		<tr>
			<td>20 &#181;L filter tips</td>
			<td>USA Scientifc Inc.</td>
			<td>P1121</td>
			<td>sterile, barrier</td>
		</tr>
		<tr>
			<td>25 mL serological pipette</td>
			<td>Corning</td>
			<td>29443-049</td>
			<td>Corning Costar Stripette individually wrapped&#160;</td>
		</tr>
		<tr>
			<td>50 mL conical tubes</td>
			<td>Corning</td>
			<td>430828</td>
			<td>graduated, sterile</td>
		</tr>
		<tr>
			<td>150 mL auto-friendly reservior</td>
			<td>Integra Bioscience</td>
			<td>6317</td>
			<td>sterile, individually wrapped for cell seeding in day 1</td>
		</tr>
		<tr>
			<td>150 mL auto-friendly reservior</td>
			<td>Integra Bioscience</td>
			<td>6318</td>
			<td>sterile, stacked, for loading dye in day 2</td>
		</tr>
		<tr>
			<td>384/ 12.5 &#181;L low retention tips</td>
			<td>Integra Bioscience</td>
			<td>6405</td>
			<td>long, sterile filter</td>
		</tr>
		<tr>
			<td>384/ 12.5 &#181;L tips</td>
			<td>Integra Bioscience</td>
			<td>6404</td>
			<td>long, sterile filter</td>
		</tr>
		<tr>
			<td>384-well plate</td>
			<td>Greiner</td>
			<td>781091</td>
			<td>CELLSTAR, clear polystyrene, &#181;Clear, Black/Flat</td>
		</tr>
		<tr>
			<td>Aluminum plate seals</td>
			<td>Axygen Scientific</td>
			<td>PCR-AS-200</td>
			<td>polyester-based</td>
		</tr>
		<tr>
			<td>Aluminum foil wrap</td>
			<td>Walmart</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Biosafty cabinet II</td>
			<td>NuAire</td>
			<td>NU-540-300</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell counter</td>
			<td>Nexcelom</td>
			<td>AutoT4</td>
			<td></td>
		</tr>
		<tr>
			<td>cell counting slides</td>
			<td>Nexcelom</td>
			<td>SD-100</td>
			<td>20 &#181;L chamber</td>
		</tr>
		<tr>
			<td>CO2 humidified incubator</td>
			<td>Thermo Fisher</td>
			<td>Forma Series II</td>
			<td></td>
		</tr>
		<tr>
			<td>Desk Lamp</td>
			<td>SunvaleeyTEK</td>
			<td>RS1000B</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide</td>
			<td>MilliporeSigma</td>
			<td>276855</td>
			<td>anhydous, &#62;99.9%</td>
		</tr>
		<tr>
			<td>Drug plate</td>
			<td>Corning</td>
			<td>3680</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s phosphate-buffered saline</td>
			<td>Corning</td>
			<td>21-031-CV</td>
			<td>DPBS, 1x without calcium amd magnesium</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Koptec</td>
			<td>2000</td>
			<td></td>
		</tr>
		<tr>
			<td>F-12K Nutrient Mixture&#160;</td>
			<td>Corning</td>
			<td>45000-354</td>
			<td>(Kaighn&#39;s Mod.) with L-glutamine</td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Equitech-Bio</td>
			<td>SFBU30</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescent calcium assay kit</td>
			<td>ENZO Lifescience</td>
			<td>ENZ-51017</td>
			<td>10x96 tests</td>
		</tr>
		<tr>
			<td>G418 sulfate</td>
			<td>Gibco Invitrogen</td>
			<td>10131-027</td>
			<td>Geneticin selective antibiotic 50 mg/mL</td>
		</tr>
		<tr>
			<td>Hank&#39;s buffer</td>
			<td>MilliporeSigma</td>
			<td>55037C</td>
			<td>HBSS modified, with calcium, with magnesium, without phenol read</td>
		</tr>
		<tr>
			<td>HEPES buffer</td>
			<td>Gibco Invitrogen</td>
			<td>15630-080</td>
			<td>1 Molar</td>
		</tr>
		<tr>
			<td>HTS data storage plateform</td>
			<td>CDD vault&#160;</td>
			<td></td>
			<td>https://www.collaborativedrug.com/</td>
		</tr>
		<tr>
			<td>Liquid handling system</td>
			<td>Integra Bioscience</td>
			<td>Viaflo</td>
			<td>384/12.5 &#181;L</td>
		</tr>
		<tr>
			<td>Plate centrifuge</td>
			<td>Thermo Fisher</td>
			<td>Sorvall ST8</td>
			<td></td>
		</tr>
		<tr>
			<td>Plate reader</td>
			<td>BMG technology</td>
			<td>Clariostar</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-D-lysine</td>
			<td>MilliporeSigma</td>
			<td>P6407</td>
			<td></td>
		</tr>
		<tr>
			<td>Rhimi-K-1 agonist peptide</td>
			<td>Genscript</td>
			<td>custom order</td>
			<td>QFSPWGamide</td>
		</tr>
		<tr>
			<td>T-75 flask</td>
			<td>Falcon</td>
			<td>353136</td>
			<td></td>
		</tr>
	</tbody>
</table>

a "dual-addition" calcium fluorescence assay for the high-throughput screening of recombinant g protein-coupled receptors

G蛋白偶联受体（GPCR）是受体的最大超家族，是许多人类药物的靶标。针对GPCR的随机小分子文库的高通量筛选（HTS）被制药行业用于靶标特异性药物发现。在这项研究中，使用HTS鉴定无脊椎动物特异性神经肽GPCRs的新型小分子配体，作为致命人类和兽医病原体载体生理研究的探针。
选择无脊椎动物特异性激肽受体作为靶标，因为它调节无脊椎动物的许多重要生理过程，包括利尿、摄食和消化。此外，许多无脊椎动物GPCR的药理学特征不佳或根本没有表征;因此，这些受体组相对于其他后生动物，特别是人类中的相关GPCR的差异药理学，为GPCR作为一个超家族的结构 - 活性关系增加了知识。为384孔板中的细胞开发了一种HTS测定法，用于从牛热蜱或南方牛蜱， Rhipicephalus microplus中发现激肽受体的配体。蜱激肽受体在CHO-K1细胞中稳定表达。
激肽受体被内源性激肽神经肽或其他小分子激动剂激活时，触发钙储存的Ca2+ 释放到细胞质中。这种钙荧光测定与“双重添加”方法相结合，可以检测同一测定板中的功能激动剂和拮抗剂“命中”分子。每次测定都是使用携带320个随机小分子阵列的药物板进行的。获得可靠的 Z' 因子为 0.7，当 HTS 处于 2 μM 终浓度时，鉴定出 3 个激动剂和 2 个拮抗剂命中分子。此处报告的钙荧光测定可以适用于筛选激活Ca2+ 信号级联的其他GPCR。

以320个随机小分子组成的内部药物板（SAC2-34-6170）为例，用于演示该HTS测定。HTS具有出色的检测质量，Z'因子为0.7（表1）。该Z'因子反映了独立于测试化合物34的测定质量。Z′因子为0.5或更大表示阳性对照和阴性对照的RFU之间的测定信号动态范围良好。Z'因子小于0.5的检测具有次优的信号动态范围，通常无法为命中选择提供信息，因此被丢弃。阳性对...

Watch this Scientific Journal Video about A "Dual-Addition" Calcium Fluorescence Assay for the High-Throughput Screening of Recombinant G Protein-Coupled Receptors at JoVE.com

A "Dual-Addition" Calcium Fluorescence Assay for the High-Throughput Screening of Recombinant G Protein-Coupled Receptors

在这项工作中，描述了一种用于384孔板的高通量细胞内钙荧光测定，以筛选重组G蛋白偶联受体（GPCR）上的小分子文库。靶标是来自牛瘟蜱的激肽受体， Rhipicephalus microplus， 在CHO-K1细胞中表达。该测定法在一个“双重添加”测定中使用相同的细胞识别激动剂和拮抗剂。

用于重组G蛋白偶联受体高通量筛选的“双加法”钙荧光测定

G protein-coupled receptors (GPCRs) represent the largest superfamily of receptors and are the targets of numerous human drugs. High-throughput screening ...

高通量筛选钙荧光双重加成测定允许鉴定通过细胞内钙级联反应发出信号的 G 蛋白偶联受体的新型小分子配体。双重加成测定的主要优点是在单次测定中检测激动剂和拮抗剂HTS，只要荧光信号持续两分钟，相同的细胞。该方法可以应用于任何通过钙级联发出信号的GPCR，其中包括大多数节肢动物神经元肽家族，即GPCR。对于测定的重现性，重要的是优化第二版已知激动剂的细胞密度、注射速度和浓度。演示该程序的将是我实验室的博士后研究助理Bianca Henriques-Santos。通过从含有70至90%汇合BMLK三个细胞的T-75烧瓶中取出用过的培养基来开始钙荧光测定。用10毫升Dulbecco的磷酸盐缓冲盐水或DPBS洗涤细胞。取出DPBS后，使用两毫升0.25%胰蛋白酶乙二胺四乙酸或EDTA分离细胞，并在37摄氏度下孵育三至五分钟。加入8毫升选择性培养基，并将细胞悬液转移到15毫升锥形管中。在以1000×g离心细胞悬液三分钟之前。弃去上清液并将细胞沉淀重悬于含有1%胎牛血清或FBS和每毫升400微克G418硫酸盐的10毫升F-12K培养基中。为了确定悬浮液的细胞密度以进一步稀释，将20微升细胞悬液混合到20微升0.4%台盼蓝中，然后将20微升混合物装入细胞计数室中，由细胞计数器读取细胞密度。使用相同的F-12K培养基稀释细胞悬液，并以密度为每毫升10至第五个细胞的四倍将最终体积补足至15毫升。将细胞接种在Poly-D赖氨酸或PDL编码的384孔板中。使用液体处理系统将25微升稀释的细胞悬液添加到板的384孔中。将板在37摄氏度和5%二氧化碳下在加湿培养箱中孵育过夜。第二天，快速倒置90%汇合的384孔板。除去用过的培养基，并在无菌纸巾上轻轻吸干两到三次，以从盘子中取出所有液体。在生物安全柜内柔和昏暗的光线下执行后续步骤。接下来，使用液体处理系统将 25 微升上样染料添加到每个孔中，方法是以每秒 3.8 微升的吸液和分液速度将 12.5 微升分液到每个孔中。重复此移液步骤，以达到每个孔中25微升的最终体积。完成后，用铝箔盖住板以保护其免受环境光照射，并在二氧化碳加湿培养箱中以 37 摄氏度孵育 30 分钟。将覆盖的板再平衡30分钟后，细胞即可进行高通量筛选或HTS。接下来，将药物板在平板离心机中以1200×g离心一分钟。将Rhimi-K-1的10x激动剂肽溶液转移到150毫升自动友好型储液器中。在酶标仪中，单击管理实验方案。然后在终点荧光强度协议中，选择Flow Forte预读，单击开始测量以测量整个HTS测定的背景信号。将细胞板插入读板器。在仪表板中，单击板上的随机孔，然后单击新的对焦高度。单击焦点调整。单击增益调整。将其指定为最大可测量荧光值的5%至10%。单击开始调整。单击开始测量以读取整个板的背景荧光信号（以相对荧光单位或 RFU）为单位。在板读数时，单击当前状态以查看实时读数值。对于双重添加测定，使用液体处理系统上下移取10微升药物溶液三次，并以每秒1.0微升的吸吸速度从药板的每个孔中吸出1.5微升。将0.5微升化合物分配到细胞测定中，以达到0.2%二甲基亚砜或DMSO中两微摩尔的最终浓度。加入筛选化合物后，立即将测定板放入酶标仪中。单击预设读取程序，沿正向和反向读取方向读取板。通过将吸头浸入含有约50毫升DPBS的150毫升废物储液罐中，处理吸头中残留的一微升药物溶液。将筛选化合物与细胞一起孵育总共五分钟，包括在生物安全柜中的读板器内熄灯的时间。接下来，使用液体处理系统使用384 12.5微升吸头将三微升激动剂肽Rhimi-K-1从储液器中加入分析板。立即将板放入读板器中。单击预设读取程序，沿正向和反向读取方向读取板。一旦完成，手动计算每个板测定质量控制的Z'因子。从在线HTS数据平台上的热图中手动选择命中分子，并计算标准化的活化百分比或NPA和抑制活性，或激动剂命中和拮抗剂命中的I0。由320个随机小分子组成的内部药物板SAC2-34-6170用于演示该HTS测定。HTS具有出色的检测质量，Z'因子为0.7，反映出检测质量与测试化合物无关。添加激动剂后立即读取板至关重要，因为荧光信号是短暂的。在此过程之后，应通过剂量反应测定验证已鉴定的命中分子，并在不表达靶GPCR的细胞中进行测试，以排除脱靶命中。

Research

JoVE Journal

Biochemistry

High-throughput Screening of Carbohydrate-degrading Enzymes Using Novel Insoluble Chromogenic Substrate Assay Kits

使用新型不溶性生色底物检测试剂盒碳水化合物降解酶的高通量筛选

Carbohydrates active enzymes (CAZymes) have multiple roles in vivo and are widely used for industrial processing in the biofuel, textile, detergent, paper ...

科研

生物化学

Method for Identifying Small Molecule Inhibitors of the Protein-protein Interaction Between HCN1 and TRIP8b

方法确定HCN1和TRIP8b之间蛋白 - 蛋白相互作用的小分子抑制剂

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are expressed ubiquitously throughout the brain, where they function to regulate the ...

Measuring G-protein-coupled Receptor Signaling via Radio-labeled GTP Binding

通过放射性标记的GTP结合测量G蛋白偶联的受体信号

G-Protein-Coupled Receptors (GPCRs) are a large family of transmembrane receptors that play critical roles in normal cellular physiology and constitute a ...

A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans

A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans

一种简单的高通量化学筛选方法在秀丽线虫中的应用

Caenorhabditis elegans is a useful organism for testing chemical effects on physiology. Whole organism small molecule screens offer significant advantages ...

A High-Throughput Luciferase Assay to Evaluate Proteolysis of the Single-Turnover Protease PCSK9

高通量荧光素酶法评价单周转蛋白酶 PCSK9 的蛋白质水解

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a single-turnover protease which regulates serum low-density lipoprotein (LDL) levels and, ...

A Semi-High-Throughput Adaptation of the NADH-Coupled ATPase Assay for Screening Small Molecule Inhibitors

用于筛选小分子抑制剂的NADH耦合ATPase分析的半高通量自适应

ATPase enzymes utilize the free energy stored in adenosine triphosphate to catalyze a wide variety of endergonic biochemical processes in vivo that would ...

High-Throughput Total Internal Reflection Fluorescence and Direct Stochastic Optical Reconstruction Microscopy Using a Photonic Chip

使用光子芯片的高通量总内反射荧光和直接随机光学重建显微镜

Total internal reflection fluorescence (TIRF) is commonly used in single molecule localization based super-resolution microscopy as it gives enhanced ...

Fully Processed Recombinant KRAS4b: Isolating and Characterizing the Farnesylated and Methylated Protein

完全加工重组KRAS4b：分离和描述脱脂和甲基化蛋白

Protein prenylation is a key modification that is responsible for targeting proteins to intracellular membranes. KRAS4b, which is mutated in 22% of human ...

Strategic Screening and Characterization of the Visual GPCR-mini-G Protein Signaling Complex for Successful Crystallization

视觉GPCR-迷你G蛋白信号复合物的战略筛选与表征，成功结晶

The key to determining crystal structures of membrane protein complexes is the quality of the sample prior to crystallization. In particular, the choice ...

Click-Chemistry Based Fluorometric Assay for Apolipoprotein N-acyltransferase from Enzyme Characterization to High-Throughput Screening

基于点击化学的载脂蛋白N-酰基转移酶荧光测定从酶表征到高通量筛选

Lipoproteins from proteobacteria are posttranslationally modified by fatty acids derived from membrane phospholipids by the action of three integral ...