Name: identifying the binding proteins of small ligands with the differential radial capillary action of ligand assay (dracala)
Uploaded: 2021-03-19T14:30:00
Description: Watch this Scientific Journal Video about Identifying the Binding Proteins of Small Ligands with the Differential Radial Capillary Action of Ligand Assay DRaCALA at JoVE.com

这项工作得到向YAZ提供的NNF项目赠款（NNF19OC0058331）以及根据玛丽·斯考多夫斯卡-居里赠款协议（No 801199）向MLS提供的欧洲联盟地平线2020研究和创新方案的支持。
<img alt="Equation 1" src="/files/ftp_upload/62331/62331img1.jpg">

1. 准备整个细胞解解剂
<ol><li>接种 大肠杆菌 K-12 ASKA ORFeome系列菌株15 到1.5mL莱索根肉汤（LB），含有25微克/mL氯霉素在96井深井板。在 30 °C 下生长 18 小时（O/N），在 160 rpm 时摇晃。第二天，在O/N培养物中加入异丙基β-d-1-硫高拉托平旁（IPTG）（最终0.5mM），以在30°C为6h时诱导蛋白质表达。</li>
	<li>颗粒细胞在 500 x g 为 10 分钟。将颗粒冻结在 -80 °C 下，直到使用。要...

<ol>
	<li>Kalia, D., 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=Nucleotide,+c-di-GMP,+c-di-AMP,+cGMP,+cAMP,+(p)ppGpp+signaling+in+bacteria+and+implications+in+pathogenesis.">Nucleotide, c-di-GMP, c-di-AMP, cGMP, cAMP, (p)ppGpp signaling in bacteria and implications in pathogenesis.</a> Chemical Society Reviews. 42 (1), 305-341 (2013).</li><li>Camilli, A., Bassler, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+small-molecule+signaling+pathways.">Bacterial small-molecule signaling pathways.</a> Science. 311 (5764), 1113-1116 (2006).</li><li>Luo, Y., 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=The+cAMP+capture+compound+mass+spectrometry+as+a+novel+tool+for+targeting+cAMP-binding+proteins:+from+protein+kinase+A+to+potassium/sodium+hyperpolarization-activated+cyclic+nucleotide-gated+channels.">The cAMP capture compound mass spectrometry as a novel tool for targeting cAMP-binding proteins: from protein kinase A to potassium/sodium hyperpolarization-activated cyclic nucleotide-gated channels.</a> Molecular &amp; Cellular Proteomics. 8 (12), 2843-2856 (2009).</li><li>Nesper, J., Reinders, A., Glatter, T., Schmidt, A., Jenal, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+capture+compound+for+the+identification+and+analysis+of+cyclic+di-GMP+binding+proteins.">A novel capture compound for the identification and analysis of cyclic di-GMP binding proteins.</a> Journal of Proteomics. 75 (15), 4874-4878 (2012).</li><li>Laventie, B. J., 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=Capture+compound+mass+spectrometry--a+powerful+tool+to+identify+novel+c-di-GMP+effector+proteins.">Capture compound mass spectrometry--a powerful tool to identify novel c-di-GMP effector proteins.</a> Journal of Visual Experiments. (97), e51404 (2015).</li><li>Roelofs, K. G., Wang, J., Sintim, H. O., Lee, V. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+radial+capillary+action+of+ligand+assay+for+high-throughput+detection+of+protein-metabolite+interactions.">Differential radial capillary action of ligand assay for high-throughput detection of protein-metabolite interactions.</a> Proceedings of the National Academy of Sciences of the United States of America. 108 (37), 15528-15533 (2011).</li><li>Zhang, Y., 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=Evolutionary+adaptation+of+the+essential+tRNA+methyltransferase+TrmD+to+the+signaling+molecule+3+',5+'-cAMP+in+bacteria.">Evolutionary adaptation of the essential tRNA methyltransferase TrmD to the signaling molecule 3 ',5 '-cAMP in bacteria.</a> Journal of Biological Chemistry. 292 (1), 313-327 (2017).</li><li>Corrigan, R. 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=Systematic+identification+of+conserved+bacterial+c-di-AMP+receptor+proteins.">Systematic identification of conserved bacterial c-di-AMP receptor proteins.</a> Proceedings of the National Academy of Sciences of the United States of America. 110 (22), 9084-9089 (2013).</li><li>Roelofs, K. G., 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=Systematic+identification+of+cyclic-di-GMP+binding+proteins+in+Vibrio+cholerae+reveals+a+novel+class+of+cyclic-di-GMP-binding+ATPases+associated+with+type+II+secretion+systems.">Systematic identification of cyclic-di-GMP binding proteins in Vibrio cholerae reveals a novel class of cyclic-di-GMP-binding ATPases associated with type II secretion systems.</a> PLoS Pathogen. 11 (10), 1005232 (2015).</li><li>Fang, X., 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=GIL,+a+new+c-di-GMP-binding+protein+domain+involved+in+regulation+of+cellulose+synthesis+in+enterobacteria.">GIL, a new c-di-GMP-binding protein domain involved in regulation of cellulose synthesis in enterobacteria.</a> Molecular Microbiology. 93 (3), 439-452 (2014).</li><li>Corrigan, R. M., Bellows, L. E., Wood, A., Grundling, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ppGpp+negatively+impacts+ribosome+assembly+affecting+growth+and+antimicrobial+tolerance+in+Gram-positive+bacteria.">ppGpp negatively impacts ribosome assembly affecting growth and antimicrobial tolerance in Gram-positive bacteria.</a> Proceedings of the National Academy of Sciences of the United States of America. 113 (12), 1710-1719 (2016).</li><li>Zhang, Y., Zbornikova, E., Rejman, D., Gerdes, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+(p)ppGpp+binding+and+metabolizing+proteins+of+Escherichia+coli.">Novel (p)ppGpp binding and metabolizing proteins of Escherichia coli.</a> Mbio. 9 (2), 02188 (2018).</li><li>Yang, J., 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=The+nucleotide+pGpp+acts+as+a+third+alarmone+in+Bacillus,+with+functions+distinct+from+those+of+(p)+ppGpp.">The nucleotide pGpp acts as a third alarmone in Bacillus, with functions distinct from those of (p) ppGpp.</a> Nature Communications. 11 (1), 5388 (2020).</li><li>Orr, M. W., Lee, V. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+radial+capillary+action+of+ligand+assay+(DRaCALA)+for+high-throughput+detection+of+protein-metabolite+interactions+in+bacteria.">Differential radial capillary action of ligand assay (DRaCALA) for high-throughput detection of protein-metabolite interactions in bacteria.</a> Methods in Molecular Biology. 1535, 25-41 (2017).</li><li>Kitagawa, 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=Complete+set+of+ORF+clones+of+Escherichia+coli+ASKA+library+(A+complete+Set+of+E.+coli+K-12+ORF+archive):+Unique+resources+for+biological+research.">Complete set of ORF clones of Escherichia coli ASKA library (A complete Set of E. coli K-12 ORF archive): Unique resources for biological research.</a> DNA Research. 12 (5), 291-299 (2005).</li><li>Hochstadt-Ozer, J., Cashel, 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+regulation+of+purine+utilization+in+bacteria.+V.+Inhibition+of+purine+phosphoribosyltransferase+activities+and+purine+uptake+in+isolated+membrane+vesicles+by+guanosine+tetraphosphate.">The regulation of purine utilization in bacteria. V. Inhibition of purine phosphoribosyltransferase activities and purine uptake in isolated membrane vesicles by guanosine tetraphosphate.</a> Journal of Biological Chemistry. 247 (21), 7067-7072 (1972).</li><li>Zhang, Y. E., 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=p)ppGpp+regulates+a+bacterial+nucleosidase+by+an+allosteric+two-domain+switch.">p)ppGpp regulates a bacterial nucleosidase by an allosteric two-domain switch.</a> Molecular Cell. 74 (6), 1239-1249 (2019).</li><li>Wang, B., 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=Affinity-based+capture+and+identification+of+protein+effectors+of+the+growth+regulator+ppGpp.">Affinity-based capture and identification of protein effectors of the growth regulator ppGpp.</a> Nature Chemical Biology. 15 (2), 141-150 (2019).</li></ol>

进行 DRACALA 筛查的关键步骤之一是获得良好的全细胞解解。首先，测试的蛋白质应大量和可溶性地生产。其次，细胞的裂解应完成，裂解剂的粘度必须极小。酶的加入和三个冷冻-解冻周期的使用往往足以完全解冻细胞。然而，释放的染色体DNA使溶质粘稠，并产生高背景结合信号，导致误报如图3B，C所示。为了减轻这种情况，可以使用来自S.马塞森的Dnase 1?...

细菌使用几个小信号分子来适应不断变化的环境1，2。例如，自动诱导剂，N-乙酰异丙胺乳酮及其改良的寡肽，调解细胞间细菌之间的交流，以协调人群行为，这种现象被称为法定人数感应2。另一组小信号分子是NSM，包括广泛研究的环状腺苷单磷酸盐（cAMP）、环二聚氨酸、环状二磷酸盐（环二磷酸盐）和瓜诺辛五磷酸盐和四磷酸盐（p）pppGpp1。细菌产生这些NSM作为对各种不同压力条件的反应。一旦产生，这些分子结合到他们的目标蛋白质，并调节几个不同的生理和代谢途径，以应付遇到的压力，提高细菌的生存能力。因此，识别目标蛋白是破译这些小分子分子功能的必然前提。
在过去的十年里，对这些小信号分子的了解激增，这主要是因为一些技术创新揭示了这些小分子的目标蛋白质。其中包括捕获复合技术3，4，5，和配体检测（DRaCALA）6的微分径向毛细管作用，本文将讨论。
DRaCALA 由文森特·李和同事于 2011年 6 月发明，它部署了硝基纤维素膜的能力，以微分隔离无蛋白质和蛋白质结合的配体。蛋白质等分子不能扩散到亚硝基纤维素膜上，而小配体（如NSM）能够扩散。通过将NSM（例如ppGpp）与要测试的蛋白质混合，并在膜上发现它们，可以预期两种情况（图1）：如果（p）ppppp与蛋白质结合，放射性标签（p）pppGpp将由蛋白质保留在点的中心，并且不会向外扩散，从而给出一个强烈的小点（即， 强烈的放射性信号）在磷构体下。然而，如果（p）pppGpp不与蛋白质结合，它将自由向外扩散，产生一个具有均匀背景放射性信号的大点。
此外，如果蛋白质存在足够的量，DRACALA可以检测小分子和整个细胞解剖中未纯化蛋白质之间的相互作用。这种简单性允许使用 DRACALA 使用 ORFeome 表达库快速识别蛋白质靶点。事实上，使用 DRaCALA 系统地识别了 cAMP7、环形二安培8、环形二聚氰化物9、10和 （p） pppGpp 11、12、13 的目标蛋白质。本视频文章以 （p） ppGpp 为例，演示和描述成功进行 DRACALA 筛查的关键步骤和考虑因素。值得注意的是，强烈建议在执行 DRACALA 之前，结合本文阅读对 DRACALA14进行更详尽的描述。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62331/62331fig01.jpg"> 图1：德拉卡拉（A）示意图分析原理。有关详细信息，请参阅文本。（B） 约束分数的量化和计算。有关详细信息，请参阅文本。简言之，DRaCALA 点将通过绘制两个圆圈来分析，这些圆圈将整个点和内部暗点（即由于测试蛋白质的结合而保留的 （p）pPpGpp）。特定的结合信号是减去非特定背景信号（按A1×计算（S 2-S 1）/（A2-A1）后内圈（S1）的放射性信号。结合分数是除以总放射性信号 （S2） 的特定绑定信号。缩写： Dracala = 利甘德分析的差速径向毛细管操作;（p） ppgpp = 瓜诺辛五角星和四磷酸盐;RT = 室温。<a href="https://www.jove.com/files/ftp_upload/62331/62331fig01large.jpg" target="_blank">请单击此处查看此图的较大版本。</a>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>32P-&#945;-GTP</td>
			<td>Perkinelmer</td>
			<td>BLU006X250UC</td>
			<td></td>
		</tr>
		<tr>
			<td>96 x pin tool</td>
			<td>V&#38;P Scientific</td>
			<td>VP 404</td>
			<td>96 Bolt Replicator, on 9 mm centers, 4.2 mm Bolt Diameter, 24 mm long</td>
		</tr>
		<tr>
			<td>96-well V-bottom microtiter plate</td>
			<td>Sterilin</td>
			<td>MIC9004</td>
			<td>Sterilin Microplate V Well 611V96</td>
		</tr>
		<tr>
			<td>Agar</td>
			<td>OXOID - Thermo Fisher</td>
			<td>LP0011</td>
			<td>Agar no. 1</td>
		</tr>
		<tr>
			<td>ASKA collection strain</td>
			<td>NBRP, SHIGEN, JAPAN</td>
			<td></td>
			<td>Ref: DNA Research, Volume 12, Issue 5, 2005, Pages 291&#8211;299. https://doi.org/10.1093/dnares/dsi012</td>
		</tr>
		<tr>
			<td>Benzonase</td>
			<td>SIGMA</td>
			<td>E1014-25KU</td>
			<td>genetically engineered endonuclease from Serratia marcescens</td>
		</tr>
		<tr>
			<td>Bradford Protein Assay Dye</td>
			<td>Bio-Rad</td>
			<td>5000006</td>
			<td>Reagent Concentrate</td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>SIGMA</td>
			<td>D8418</td>
			<td>&#8805;99.9%</td>
		</tr>
		<tr>
			<td>DNase 1</td>
			<td>SIGMA</td>
			<td>DN25-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>gel filtration10x300 column</td>
			<td>GE Healthcare</td>
			<td>28990944</td>
			<td>contains 20% ethanol as preservative</td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>PanReac AppliChem</td>
			<td>122329.1214</td>
			<td>Glycerol 87% for analysis</td>
		</tr>
		<tr>
			<td>Hypercassette</td>
			<td>Amersham</td>
			<td>RPN 11647</td>
			<td>20 x 40 cm</td>
		</tr>
		<tr>
			<td>Imidazole</td>
			<td>SIGMA</td>
			<td>56750</td>
			<td>puriss. p.a., &#8805; 99.5% (GC)</td>
		</tr>
		<tr>
			<td>IP Storage Phosphor Screen</td>
			<td>FUJIFILM</td>
			<td>28956474</td>
			<td>BAS-MS 2040 20x 40 cm</td>
		</tr>
		<tr>
			<td>Isopropyl &#946;-d-1-thiogalactopyranoside (IPTG)</td>
			<td>SIGMA</td>
			<td>I6758</td>
			<td>Isopropyl &#946;-D-thiogalactoside</td>
		</tr>
		<tr>
			<td>Lysogeny Broth (LB)</td>
			<td>Invitrogen - Thermo Fisher</td>
			<td>12795027</td>
			<td>Miller&#39;s LB Broth Base</td>
		</tr>
		<tr>
			<td>Lysozyme</td>
			<td>SIGMA</td>
			<td>L4949</td>
			<td>from chicken egg white; BioUltra, lyophilized powder, &#8805;98%</td>
		</tr>
		<tr>
			<td>MgCl2 (Magnesium chloride)</td>
			<td>SIGMA</td>
			<td>208337</td>
			<td></td>
		</tr>
		<tr>
			<td>MilliQ water</td>
			<td></td>
			<td></td>
			<td>ultrapure water</td>
		</tr>
		<tr>
			<td>multichannel pipette</td>
			<td>Thermo Scientific</td>
			<td>4661110</td>
			<td>F1 - Clip Tip; 1-10 ul, 8 x channels</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>VWR Chemicals</td>
			<td>27810</td>
			<td>AnalaR NORMAPUR, ACS, Reag. Ph. Eur.</td>
		</tr>
		<tr>
			<td>Ni-NTA Agarose</td>
			<td>Qiagen</td>
			<td>30230</td>
			<td></td>
		</tr>
		<tr>
			<td>Nitrocellulose Blotting Membrane</td>
			<td>Amersham Protran</td>
			<td>10600003</td>
			<td>Premium 0.45 um 300 mm x 4 m</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>OXOID - Thermo Fisher</td>
			<td>BR0014G</td>
			<td>Phosphate buffered saline (Dulbecco A), Tablets</td>
		</tr>
		<tr>
			<td>PEG3350 (Polyethylene glycol 3350)</td>
			<td>SIGMA</td>
			<td>202444</td>
			<td></td>
		</tr>
		<tr>
			<td>phenylmethylsulfonyl fluoride (PMSF)</td>
			<td>SIGMA</td>
			<td>93482</td>
			<td>Phenylmethanesulfonyl fluoride solution - 0.1 M in ethanol (T)</td>
		</tr>
		<tr>
			<td>Phosphor-imager</td>
			<td>GE Healthcare</td>
			<td>28955809</td>
			<td>Typhoon FLA-7000 Phosphor-imager</td>
		</tr>
		<tr>
			<td>Pipette Tips, filtered</td>
			<td>Thermo Scientific</td>
			<td>94410040</td>
			<td>ClipTip 12.5 &#956;l nonsterile</td>
		</tr>
		<tr>
			<td>Poly-Prep Chromatography column</td>
			<td>Bio-Rad</td>
			<td>7311550</td>
			<td>polypropylene chromatography column</td>
		</tr>
		<tr>
			<td>Protease inhibitor Mini</td>
			<td>Pierce</td>
			<td>A32955</td>
			<td>Tablets, EDTA-free</td>
		</tr>
		<tr>
			<td>screw cap tube</td>
			<td>Thermo Scientific</td>
			<td>3488</td>
			<td>Microcentifuge Tubes, 2.0 ml with screw cap, nonsterile</td>
		</tr>
		<tr>
			<td>SLS 96-deep Well plates</td>
			<td>Greiner</td>
			<td>780285</td>
			<td>MASTERBLOCK, 2 ML, PP, V-Bottom, Natural</td>
		</tr>
		<tr>
			<td>spin column</td>
			<td>Millipore</td>
			<td>UFC500396</td>
			<td>Amicon Ultra -0.5 ml Centrifugal Filters</td>
		</tr>
		<tr>
			<td>Thermomixer</td>
			<td>Eppendorf</td>
			<td>5382000015</td>
			<td>Thermomixer C</td>
		</tr>
		<tr>
			<td>TLC plate (PEI-cellulose F TLC plates)</td>
			<td>Merck Millipore</td>
			<td>105579</td>
			<td>DC PEI-cellulose F (20 x 20 cm)</td>
		</tr>
		<tr>
			<td>Tris</td>
			<td>SIGMA</td>
			<td>BP152</td>
			<td>Tris Base for Molecular Biology</td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>SIGMA</td>
			<td>P1379</td>
			<td>viscous non-ionic detergent</td>
		</tr>
		<tr>
			<td>&#946;-mercaptoethanol</td>
			<td>SIGMA</td>
			<td>M3148</td>
			<td>99% (GC/titration)</td>
		</tr>
	</tbody>
</table>

identifying the binding proteins of small ligands with the differential radial capillary action of ligand assay (dracala)

在过去的十年里，细菌生理学中对小信号分子的理解取得了巨大的进步。特别是，在模型生物体中系统地识别和研究了几个核苷酸衍生的二次信使（NSM）的目标蛋白质。这些成就主要归功于一些新技术的发展，包括捕获复合技术和配体检测（DRaCALA）的微分径向毛细管作用，这些技术被用来系统地识别这些小分子的目标蛋白质。本文描述了使用NSM，瓜诺辛五角星和四磷酸盐（p）pppGpp，作为DRACALA技术的例子和视频演示。使用DRaCALA，在模型有机体 Escherichia大肠杆菌 K-12中发现了20种已知蛋白质中的9种，以及12种新靶蛋白（p）pppGpp，显示了这种测定的力量。原则上，DRACALA可用于研究可标有放射性同位素或荧光染料的小配体。这里讨论了 DRACALA 的关键步骤、优点和缺点，以便进一步应用此技术。

遵循上述协议通常会产生两种类型的结果（图3）。
图 3A显示大多数油井的背景绑定信号相对较低的板（绑定分数< 0.025）。来自油井 H3 的正绑定信号给出的绑定分数为 ±0.35，远远高于其他油井的绑定分数。即使没有量化，H3 也非常显著，表明 H3 中表达的目标蛋白质与 pppGpp、ppGpp 或两者兼有。事实上，在H3井中过度表达的蛋白质是...

Watch this Scientific Journal Video about Identifying the Binding Proteins of Small Ligands with the Differential Radial Capillary Action of Ligand Assay DRaCALA at JoVE.com

Identifying the Binding Proteins of Small Ligands with the Differential Radial Capillary Action of Ligand Assay DRaCALA

利甘德分析（DRaCALA）的微分径向毛细管作用可用于使用ORFeome库识别生物体的小配体结合蛋白。

用利甘德分析（DRACALA）的微分径向毛细管作用识别小配体的结合蛋白

The past decade has seen tremendous progress in the understanding of small signaling molecules in bacterial physiology. In particular, the target proteins ...

小信号分子瞄准各种效应蛋白，以控制细菌毒性和人类生物学。然而，这些效应蛋白是具有挑战性的识别。作为系统生物学工具，DRaCALA 允许使用 OVium 库对未知效应物蛋白进行可行、快速和高度敏感的识别。DRACALA可用于研究任何小型信号分子，只要它可以标有放射性同位素或荧光染料。首先将大肠杆菌接种到1.5毫升的LB中，每米96井深井板中加入25微克氯霉素，在30摄氏度和每分钟160次旋转下进行隔夜孵育。第二天早上，在30摄氏度下用500微摩尔IPTG治疗通宵培养物6小时，以诱导蛋白质表达。在孵化结束时，通过离心将细胞颗粒化，取出超高分子，并将样品冻结在零下80摄氏度。要启动裂解，在每口井150微升裂解缓冲器中重新使用颗粒，在零下80摄氏度下孵育30分钟，然后在37摄氏度下解冻细胞20分钟。然后，解酯应储存在零下80摄氏度，然后使用。要完成过度表达蛋白质的细胞解析，将样品重新浸入40毫升冰冷裂解缓冲器2中，在2秒内以60%的振幅对样品进行声波处理，4秒后8分钟，然后通过离心清除裂解物。当样品被离心时，在站立的聚丙烯色谱柱中加入500微升同质镍-NTA树脂。15分钟后，用15毫升超纯水洗两次安定树脂，用15毫升裂解缓冲器洗两次。在离心机结束时，将清除的解糖超高分子加载到柱子上。当所有的裂解液都流过柱子时，用30毫升的洗涤缓冲器清洗柱子。为了使蛋白质变质，在柱子上加入400微升的弹性缓冲液，并重复三次。再用 300 微升体积的缓冲器重复三次，并将精化蛋白组合在 700 微升的最后体积中。对于精制样品的凝胶过滤，在用 25 毫升新鲜制备的凝胶过滤缓冲器清洗尺寸排除列后，使用 500 微升环将整个体积的 elt 蛋白加载到柱子上。以每分钟 500 微升的速度运行，收集两到三个 500 微升体积的相应蛋白质分数。然后使用单个自旋柱浓缩每个蛋白质，并使用布拉德福德检测套件根据标准协议测量蛋白质浓度。合成标有瓜诺辛五磷酸盐的磷32，将小规模的Relseq反应组装在桌子上勾勒出的螺丝盖管中，在37摄氏度的热混合器中孵育反应一小时，在95摄氏度下孵化5分钟，然后在冰上孵化5分钟。在孵化结束时，通过离心将沉淀蛋白旋转下来，并将含有超高磷酸盐的合成磷32标记为瓜诺辛五磷酸盐转移到一个新的螺丝盖管中。对于磷32瓜诺辛四磷酸盐合成，加入一个微摩尔GppA到磷32标签的五磷酸盐产品的一半在新的螺丝盖管和孵育反应10分钟在37摄氏度，5分钟在95摄氏度，5分钟在冰上。在孵化结束时，通过离心将沉淀物旋转，并将标有瓜诺辛四磷酸盐的磷32标记到一个新的管子中。要分析分离的目标蛋白，使用 1.5 摩尔单磷酸盐作为移动相，在薄层色谱板上运行每个样品的一微升。分析后，将干燥板放入透明塑料文件夹中，将板暴露在存储磷屏上五分钟，然后可视化和量化磷成像仪上的数据。对于目标蛋白质的 DRaCALA 筛选，在 95 井 V 底微蒂板的个别井中加入 20 微升解冻批发裂解剂，并在每口油井中加入 2.5 单位的塞拉蒂亚马塞森内脏。在37摄氏度下15分钟后，将解冻剂放在冰上20分钟。接下来，混合等量的磷32标记的瓜诺辛五磷酸盐和瓜诺辛四磷酸盐，并添加1X裂解缓冲器一到混合物，以获得四纳米摩尔瓜诺辛五磷酸盐溶液。使用多通道移液器和过滤移液器尖端，将瓜诺辛五磷酸盐混合物的10微升与细胞裂解液混合，在室温下孵育5分钟。在孵化结束时，在 0.01% 的非离子洗涤剂溶液中清洗 96X 针脚工具三次，然后每次洗涤在纸巾上干燥 30 秒，然后将针脚工具放入 96 井样品板中。30 秒后，将销工具直接抬起，将其直接放在硝基纤维素膜上 30 秒。干燥五分钟后，将硝基纤维素膜放入透明塑料文件夹中，以便通过磷成像进行存储磷屏暴露和可视化。要量化和识别潜在的目标蛋白，在与磷成像仪相关的分析软件中，打开可视化板的凝胶文件。要定义要分析的点，请使用阵列分析功能设置 12 列乘 8 行网格。要限制孔点的外缘，定义大圆，将定义的大圆的体积加背景和面积导出到电子表格中。要限制小内点，缩小定义的圆圈，导出定义的小圆的体积加背景和区域，并保存电子表格中的所有数据。位置圆圈在必要时与点重叠，调整比实际点稍大的大小。使用方程计算电子表格中的绑定分数并绘制数据图。然后识别与其他大多数油井相比，油井中具有高结合分数的潜在结合蛋白。在此具有代表性的分析中，可以观察到大多数油井中背景绑定信号相对较低的板。由于瓜诺辛五磷酸盐结合蛋白Hpt的过度表达，H3井中的正结合信号表现出的结合分数远远高于其他油井的结合分数。在代表性板块中，几口井显示出相对较高的背景结合信号，如许多油井中观察到的相对强的内点，以及量化后观察到的一贯高结合分数。值得注意的是，一些目标还给出了可变的绑定分数，例如误报。为了进一步澄清，研究人员使用纯化蛋白再次测试结合。一旦目标蛋白质被识别出来，人们就可以用 DRACALA 、同温热度或其他方法将它们纯化为同质性，并确认相互作用强度和特异性。识别小信号分子的目标蛋白质为深入了解细菌毒性对人类生物学的作用铺平了道路。

Research

JoVE Journal

Biochemistry

Identifying the Binding Proteins of Small Ligands with the Differential Radial Capillary Action of Ligand Assay (DRaCALA)

Identification of Small Molecule-binding Proteins in a Native Cellular Environment by Live-cell Photoaffinity Labeling

小分子结合蛋白的鉴定在天然细胞环境由活细胞光亲和标记

Identifying the molecular target(s) of small molecules is a challenging but necessary step towards understanding their mechanism of action. While several ...

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 Biomolecular DSC Profiles with Thermolabile Ligands to Rapidly Characterize Folding and Binding Interactions

用耐热配体测量生物分子 DSC 剖面快速表征折叠和结合的相互作用

Differential scanning calorimetry (DSC) is a powerful technique for quantifying thermodynamic parameters governing biomolecular folding and binding ...

Nitrogen Cavitation and Differential Centrifugation Allows for Monitoring the Distribution of Peripheral Membrane Proteins in Cultured Cells

氮气气蚀和差速离心允许监测外周血膜蛋白在细胞中的分布

Cultured cells are useful for studying the subcellular distribution of proteins, including peripheral membrane proteins. Genetically encoded fluorescently ...

Rab10 Phosphorylation Detection by LRRK2 Activity Using SDS-PAGE with a Phosphate-binding Tag

用磷酸盐结合标记的 SDS-页 Rab10 磷酸化检测 LRRK2 活性

Mutations in leucine-rich repeat kinase 2 (LRRK2) have been shown to be linked with familial Parkinson&#39;s disease (FPD). Since abnormal activation of ...

Capturing the Interaction Kinetics of an Ion Channel Protein with Small Molecules by the Bio-layer Interferometry Assay

用生物层干涉法捕获小分子离子通道蛋白的相互作用动力学

The bio-layer interferometry (BLI) assay is a valuable tool for measuring protein-protein and protein-small molecule interactions. Here, we first describe ...

Method for Efficient Refolding and Purification of Chemoreceptor Ligand Binding Domain

化学感受器配体结合域的高效复用与纯化方法

Identification of natural ligands of chemoreceptors and structural studies aimed at elucidation of the molecular basis of the ligand specificity can be ...

Detection of Small GTPase Prenylation and GTP Binding Using Membrane Fractionation and  GTPase-linked Immunosorbent Assay

膜分馏和 gtpase-关联免疫吸附法检测小 gtpase 预基化和 gtp 结合

The Rho GTPase family belongs to the Ras superfamily and includes approximately 20 members in humans. Rho GTPases are important in the regulation of ...

Analysis of AtHIRD11 Intrinsic Disorder and Binding Towards Metal Ions by Capillary Gel Electrophoresis and Affinity Capillary Electrophoresis

毛细管电泳与亲和毛细管电泳对金属离子 AtHIRD11 固有紊乱与结合的分析

Plants are strongly dependent on their environment. In order to adjust to stressful changes (e.g., drought and high salinity), higher plants evolve ...

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 ...