1.肽序列稀释<ol><li>制备测定缓冲液:汉克斯缓冲补充有25mM的HEPES，0.1％BSA和0.5mM 3-异丁基-1-甲基黄嘌呤（IBMX），pH 7.4的盐溶液（HBSS）。 </li><li>使用批量试剂分配器5微升测定缓冲液系统添加到每个孔5 384孔低体积试验平板。 <ol><li>使用内部软件以5微升体积除一个384孔平板的每一个以及每个制造商的说明建立的分配程序。 </li><li>沉浸在分析缓冲液和总理点胶盒管。 </li><li>放置384孔板载体上的低体积试验板。 </li><li>按开始。 </li></ol></li><li>冲淡一切肽样品，无论存储车辆，在测定缓冲液产生100倍的股票肽。 注意:需要筛选肽可在磷酸盐缓冲盐水（PBS）或二甲基亚砜（DMSO），为被认为适当的（如，DMSO瓦特提供病有与二级修饰如聚乙二醇化）的肽具有有害影响。 </li><li>免除25微升100X肽股成列的声学合格384孔微孔板聚丙烯1-5。该板指定"源板"。确保源板，而不是目的板，是平底，并符合特定声公差由电声乐器的制造商定义。 </li><li>免除25微升100X参考控制入井A23和源板A的A24 </li><li>将10微升检测缓冲液到列11-15和30微升检测缓冲液到源盘A的21-22列</li><li>将10微升分析缓冲液为列6-10和16-20列第二声学合格的384孔微孔板聚丙烯的。该板指定"源板B&#39;。 </li><li>离心机源板A和B在300 XG 1分钟。包括适当的平衡板。 </li><li>使用声fluið分配器集成到自动化机器人系统，制备三个顺序1:从第1列中的源A的100倍的肽股100中间稀释液（在测定缓冲液） 注:编程需要重新格式化板和（由声学流体分配器的制造商提供）剂量反应软件允许源A和源B（参见图1板布局）之间的三声传输。步骤1.9特性的细节运用在受自动机械手控制这个实验室使用的液体分配器（见材料表 ）: <ol><li>载源板和检测板成机器人的板酒店部分。 </li><li>开放式自动化机器人的软件和负载板格式化和剂量反应程序扩展协议。点击"运行"。 注:自动化指示声流体分配器从源头板列1-5转移250 NL到源盘B 6-10列和第二ACOUSTIç流体分配器能够处理更大的液体体积与15微升分析缓冲液混合回填。然后自动化源盘B传送到集成离心机在300 XG 1分钟离心机（一种适当的平衡板是包括所有的离心步骤）。源板B被返回到声学流体分配器250 NL的转移从源盘B的6-10列到源板A的11-15列和用15微升测定缓冲回填混合。自动化传输源板，以集成离心机在300 XG 1分钟离心机。然后源板A被返回到声学流体分配器250 NL的转移从源盘A的11-15列到源盘B的16-20列和用15微升测定缓冲回填混合。自动化然后在300 XG 1分钟源盘B传送到集成离心机和离心机。 注:最后剂量响应协议指示声学分配传送从各4连续稀释源板孔中所需的体积（在源板A和源盘B两者）来构造在测定板一式两份的完整的11点曲线（预填充有5微升测定缓冲液在上面的步骤1.2 ）。自动化转移测定板，以集成离心机和离心机在300×g离心1分钟。 </li></ol></li></ol> 2.细胞的制备<ol><li>在20毫升测定缓冲液37ºC水浴悬浮迅速表达靶小鼠GLP-1受体解冻冷冻保存的中国仓鼠卵巢（CHO）细胞。 </li><li>离心细胞悬浮液，在室温下（RT）200 xg离心5分钟。包括适当的平衡。 </li><li>弃上清，重悬在10毫升分析缓冲液细胞沉淀。 </li><li>稀释细胞库存1:1台盼蓝和使用自动细胞计数器测定活细胞密度。悬浮细胞以每毫升1.6×10 6个细胞测定缓冲液（相当于每逢8000细胞测定板的1）。 </li><li>使用批量试剂分配器5微升细胞悬液加入到测定板（含于5μl测定缓冲液连续稀释的肽）的每个孔中，并孵育在室温30分钟。 </li></ol> 3. HTRF cAMP的检测分析<ol><li>带来HTRF cAMP测定试剂盒RT下在使用前30分钟。 </li><li>准备每个HTRF试剂（穴和D2）分别在裂解液1:20稀释（专利配方，通过cAMP的检测化验的制造商提供）。 注意:注意:裂解液中含有氟化钾（KF），这是有毒的致畸胎13。有关处理，含有KF检测板应密封并焚烧，任何液体废物处置，必须顺着水槽之前，稀释&lt;1 mmol / L的水。 </li><li>使用批量试剂分配器5微升穴试剂加入到测定板的所有孔中。 </li><li>使用批量试剂分配器5微升D2试剂加入到柱纳秒测定板1-22。 </li><li>立即手动移液5微升裂解缓冲液进入测定板的孔A23-D24，得到非特异性结合（NSB）对照孔，和5μld2的试剂的成孔E23-P24。 </li><li>离心机检测板1分钟，在室温200 XG混合井。 </li><li>覆盖试验板，以减少蒸发和光致漂白，并在室温孵育1小时。 </li><li>用在320nm处激发和发射在620nm，并使用板读数器665纳米测量荧光共振能量转移（FRET）信号。 </li></ol> 4.数据分析<ol><li>使用平均NSB从所有孔减去背景。 </li><li>从665纳米/ 620纳米比率计算Δ％˚F如下: 比率=（A 665nm / A 620nm处 ）×10 4 三角洲F =（（采样率-比NSB）/比例NSB））×100 其中，比例NSB =没有D2试剂孔。 </li><li>计算％ACTIVAT未刺激的细胞与细胞之间的离子与最大GLP-1的配体刺激的如下: ％活化=（Δ％FSAMPLE - ％DeltaFunstimulated细胞）/（％DeltaFGLP-1刺激的细胞 - ％DeltaF未刺激细胞）×100 </li><li>通过参考控制1所限定分析通过4参数后勤分析;图作为％活化浓度-响应曲线。 </li></ol>

<ol>
	<li>Naylor, J., Rossi, A., Hornigold, 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=Acoustic+Dispensing+Preserves+the+Potency+of+Therapeutic+Peptides+throughout+the+Entire+Drug+Discovery+Workflow.">Acoustic Dispensing Preserves the Potency of Therapeutic Peptides throughout the Entire Drug Discovery Workflow.</a> J.Lab.Autom. 21 (1), 90-96 (2016).</li><li>Campbell, J. E., Drucker, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacology,+physiology,+and+mechanisms+of+incretin+hormone+action.">Pharmacology, physiology, and mechanisms of incretin hormone action.</a> Cell.Metab. 17 (6), 819-837 (2013).</li><li>Hui, H., Farilla, L., Merkel, P., Perfetti, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+short+half-life+of+glucagon-like+peptide-1+in+plasma+does+not+reflect+its+long-lasting+beneficial+effects.">The short half-life of glucagon-like peptide-1 in plasma does not reflect its long-lasting beneficial effects.</a> Eur.J.Endocrinol. 146 (6), 863-869 (2002).</li><li>Harris, D., Olechno, J., Datwani, S., Ellson, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gradient,+contact-free+volume+transfers+minimize+compound+loss+in+dose-response+experiments.">Gradient, contact-free volume transfers minimize compound loss in dose-response experiments.</a> J.Biomol.Screen. 15 (1), 86-94 (2010).</li><li>Ekins, S., Olechno, J., Williams, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dispensing+Processes+Impact+Apparent+Biological+Activity+as+Determined+by+Computational+and+Statistical+Analyses.">Dispensing Processes Impact Apparent Biological Activity as Determined by Computational and Statistical Analyses.</a> PLoS ONE. 8 (5), 62325 (2013).</li><li>Goebel-Stengel, M., Stengel, A., Tache, Y., Reeve, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+importance+of+using+the+optimal+plasticware+and+glassware+in+studies+involving+peptides.">The importance of using the optimal plasticware and glassware in studies involving peptides.</a> Anal.Biochem. 414 (1), 38-46 (2011).</li><li>McDonald, G. R., 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=Bioactive+Contaminants+Leach+from+Disposable+Laboratory+Plasticware.">Bioactive Contaminants Leach from Disposable Laboratory Plasticware.</a> Science. 322 (5903), 917 (2008).</li><li>Belaiche, C., Holt, A., Saada, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonylphenol+ethoxylate+plastic+additives+inhibit+mitochondrial+respiratory+chain+complex+I.">Nonylphenol ethoxylate plastic additives inhibit mitochondrial respiratory chain complex I.</a> Clin Chem. 55 (10), 1883-1884 (2009).</li><li>Sackmann, E. K., 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=Technologies+That+Enable+Accurate+and+Precise+Nano-+to+Milliliter-Scale+Liquid+Dispensing+of+Aqueous+Reagents+Using+Acoustic+Droplet+Ejection.">Technologies That Enable Accurate and Precise Nano- to Milliliter-Scale Liquid Dispensing of Aqueous Reagents Using Acoustic Droplet Ejection.</a> J.Lab.Autom. 21 (1), 166-177 (2016).</li><li>Grant, R. 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=Achieving+accurate+compound+concentration+in+cell-based+screening:+validation+of+acoustic+droplet+ejection+technology.">Achieving accurate compound concentration in cell-based screening: validation of acoustic droplet ejection technology.</a> J.Biomol.Screen. 14 (5), 452-459 (2009).</li><li>Turmel, M., Itkin, Z., Liu, D., Nie, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+Innovative+Way+to+Create+Assay+Ready+Plates+for+Concentration+Response+Testing+Using+Acoustic+Technology.">An Innovative Way to Create Assay Ready Plates for Concentration Response Testing Using Acoustic Technology.</a> J.Lab.Autom. 15 (4), 297-305 (2010).</li><li>Butler, R., 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=Use+of+the+site-specific+retargeting+jump-in+platform+cell+line+to+support+biologic+drug+discovery.">Use of the site-specific retargeting jump-in platform cell line to support biologic drug discovery.</a> J.Biomol.Screen. 20 (4), 528-535 (2015).</li><li>Panchal, S., Verma, 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=Effect+of+sodium+fluoride+in+maternal+and+offspring+rats+and+its+amelioration.">Effect of sodium fluoride in maternal and offspring rats and its amelioration.</a> Asian Pac.J.Reprod. 3 (1), 71-76 (2014).</li><li>Hanson, S. M., Ekins, S., Chodera, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+error+in+experimental+assays+using+the+bootstrap+principle:+understanding+discrepancies+between+assays+using+different+dispensing+technologies.">Modeling error in experimental assays using the bootstrap principle: understanding discrepancies between assays using different dispensing technologies.</a> J.Comput. Aided Mol.Des. 29 (12), 1073-1086 (2015).</li><li>Harris, D., Olechno, J., Datwani, S., Ellson, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gradient,+Contact-Free+Volume+Transfers+Minimize+Compound+Loss+in+Dose-Response+Experiments.">Gradient, Contact-Free Volume Transfers Minimize Compound Loss in Dose-Response Experiments.</a> J.Biomol. Screen. 15 (1), 86-94 (2010).</li><li>Chan, G. K. Y., Wilson, S., Schmidt, S., Moffat, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unlocking+the+potential+of+high-throughput+drug+combination+assays+using+acoustic+dispensing.">Unlocking the potential of high-throughput drug combination assays using acoustic dispensing.</a> J.Lab.Autom. 21 (1), 125-132 (2016).</li><li>Roberts, K., 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=Implementation+and+challenges+of+direct+acoustic+dosing+into+cell-based+assays.">Implementation and challenges of direct acoustic dosing into cell-based assays.</a> J.Lab.Autom. 21 (1), 76-89 (2016).</li></ol>

The authors have nothing to disclose.

这个协议描述自动声学分配的成功应用到在3×10 6个要求小于1微升样品的浓度范围连续稀释的肽样品。这种方法的主要优点是通过经由还原样品暴露于所通常所需的试剂转移和混合试验容器和塑料制品（如枪头）最小化的肽吸附到塑料制品，以提高数据质量。虽然声学分配不能完全排除使用塑料制品的，它确实显著酌减。此外，消除基于尖部-移液还消除了样品的交叉污染，和错误不会在?...

针对GLP-1R是2型糖尿病2的治疗已建立的药物靶标。对于该受体的天然肽激动剂，GLP-1，具有2-3分钟3 的体内半衰期。 GLP-1的其G蛋白偶联靶受体的结果在下游生产通过天然G蛋白偶联的第二信使cAMP对腺苷酸环化酶的活化的结合。累积的cAMP测量提供了可靠的测定法来监测受体激活和筛选活性GLP-1类似物与优选的物理化学性质。这样的测定需要测试样品的连续稀释来构建浓度 - 响应曲线，而这移交肽样品时，是特别复杂。从基于尖连续稀释准备的潜在错误先前已经1,4,5描述。肽将吸附到塑料制品，造成不可靠的效力的估计。肽损失，可以通过T为最小化他纳入缓冲器牛血清白蛋白（BSA）和使用硅化塑料制品的，但蛋白质结合的仍无法预测。特别是，在实验容器GLP-1的结合的变体已经描述6。有在实验室塑料制品中使用可从提示和微量滴定板成水测定缓冲液浸出，并与蛋白质功能7,8干扰该稳定剂进一步复杂化。因此，方法，以减少暴露于塑料制品是必要的，以增加测量的准确度。 声学液体分配器集中的高频声信号到流体样品的表面上，从而导致在精确纳升液滴喷射到相邻测定板9。利用声学弹射的是制药行业的准备和大量的合成化合物库的筛选标准，而且技术已经很好的验证了小moleculES 10。据我们所知，我们是来形容声配药重组和合成肽的制备第一组和我们先前已经报道了更高的精度比传统的基于技巧的方法1。 本文介绍了在完全自动化的处理板的机器人系统由非接触式声学传递肽序列和直接稀释配制的整合。许多围绕样本声传输方法先前已被描述11。我们利用两步法来制备中间库存浓度和以串行稀释肽类似物为全剂量反应曲线的产生。制备的肽温育的细胞表达靶小鼠GLP-1R，并且我们使用市售均相时间分辨荧光（HTRF）测定法来测量这些细胞中的cAMP积累作为肽激动剂ACTIV的读出性。该测定是健壮和适合于高通量384孔格式和常规应用到分析开发和药物筛选项目12。

<table border="0" cellpadding="0" cellspacing="0" width="1339">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:469px;">Hanks&#8217; Balanced Salt solution</td>
			<td style="width:235px;">Sigma-Aldrich</td>
			<td style="width:119px;">H8264</td>
			<td style="width:517px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:469px;">HEPES</td>
			<td style="width:235px;">Sigma-Aldrich</td>
			<td style="width:119px;">H3375</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:469px;">Bovine Serum Albumin</td>
			<td style="width:235px;">Sigma-Aldrich</td>
			<td style="width:119px;">A9418</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:469px;">3-Isobutyl-1-methylxanthine</td>
			<td style="width:235px;">Sigma-Aldrich</td>
			<td style="width:119px;">I7018</td>
			<td>Prepared as a 0.5 M stock in DMSO</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:469px;">GLP-1 (7-36) amide</td>
			<td style="width:235px;">Bachem</td>
			<td>H-6795</td>
			<td>Prepared as a 1 mg/ml stock in PBS, referred to as &#39;100X reference control&#39;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:469px;">Test peptides</td>
			<td style="width:235px;">Produced in-house at MedImmune</td>
			<td></td>
			<td>Supplied at various concentrations in DMSO or PBS as appropriate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:469px;">100X peptide stock</td>
			<td style="width:235px;">Produced in-house at MedImmune</td>
			<td></td>
			<td>Test peptide diluted into assay buffer to 100X final required concentration</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:469px;">Trypan Blue Solution, 0.4%</td>
			<td style="width:235px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">15250-061</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:469px;">Cedex XS Cell Analyzer</td>
			<td style="width:235px;">Innovatis</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:469px;">Corning 384 well plates, low volume</td>
			<td style="width:235px;">Sigma-Aldrich</td>
			<td style="width:119px;">4514</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:469px;">Echo Qualified 384-Well Polypropylene Microplate</td>
			<td style="width:235px;">Labcyte Inc.</td>
			<td style="width:119px;">P-05525</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:469px;">Echo Qualified Reservoir</td>
			<td style="width:235px;">Labcyte Inc.</td>
			<td style="width:119px;">ER-0055</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:469px;">Echo 550 Liquid Handler</td>
			<td style="width:235px;">Labcyte Inc.</td>
			<td style="width:119px;"></td>
			<td>Droplet transfer volumes in increments of 2.5 nl</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:469px;">Echo 525 Liquid Handler</td>
			<td style="width:235px;">Labcyte Inc.</td>
			<td style="width:119px;"></td>
			<td>Droplet transfer volumes in increments of 25 nl</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:469px;">ACell Benchtop Automation&#160;</td>
			<td style="width:235px;">HighRes Biosolutions</td>
			<td style="width:119px;">MC522</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:469px;">Cellario Lab Automation Scheduling software for Life Science Robotics</td>
			<td style="width:235px;">HighRes Biosolutions</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:469px;">MultidropCombi Reagent Dispenser</td>
			<td style="width:235px;">ThermFisher Scientific</td>
			<td style="width:119px;">5840300</td>
			<td>Referred to as &#39;bulk reagent dispenser&#39;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:469px;">HTRF cAMP Dynamic 2 kit</td>
			<td style="width:235px;">Cisbio Bioassays</td>
			<td style="width:119px;">62AM4PEJ</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:469px;">EnVision Multilabel Reader</td>
			<td style="width:235px;">PerkinElmer</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

automated acoustic dispensing for the serial dilution of peptide agonists in potency determination assays

与小分子药物的发现，筛选肽激动剂要求的肽，以产生浓度 - 响应曲线的连续稀释。筛选的肽，得到复杂的附加层作为常规的基于针尖 - 样品处理方法暴露肽塑料制品的一个大的表面面积，提供用于通过吸附肽损失增加的机会。防止过度暴露于塑料制品通过加入塑料降低肽损失，因而在效价的预测最小误差，我们先前已经描述了非接触式的声学分配用于肽激动剂1的体外高通量筛选的好处。这里我们讨论了在利用在小鼠胰高血糖素样肽1受体（GLP-1R）筛选肽激动剂的例子中微滴定板的肽系列稀释的非接触声学制备一个完全集成的自动化解决方案。我们的方法允许高-throughput细胞为基础的分析来筛选激动剂，很容易扩展到支持增加的样品通量，或允许检测板拷贝数量增加（ 例如 ，对于更多的靶细胞系面板）。

<html>我们经常使用两步法通过声传递稀释肽。用于第一步骤中，用自动化对准的声学分配装置被用来创建四个库存肽跨两个源板中间稀释液（ 图1a，b） 中 。对于第二个步骤中，我们使用的声分配器从源板A和B，以进一步稀释库存稀释创建为每个测试肽（ 图1c）11点浓度范围。每种肽的浓度范围是在重复烧制，左跨越测定?...

Watch this Scientific Journal Video about Automated Acoustic Dispensing for the Serial Dilution of Peptide Agonists in Potency Determination Assays at JoVE.com

Automated Acoustic Dispensing for the Serial Dilution of Peptide Agonists in Potency Determination Assays

Peptide adsorption to plasticware during traditional tip-based serial dilutions can significantly impact potency determination and confound the understanding of structure-activity relationships used for lead identification and lead optimization phases of drug discovery. Here methods for automated acoustic non-contact serial dilution of peptide samples are described.

自动声胶肽受体激动剂的效价测定测定的连续稀释

As with small molecule drug discovery, screening for peptide agonists requires the serial dilution of peptides to produce concentration-response curves. ...

automated-acoustic-dispensing-for-serial-dilution-peptide-agonists

Research

JoVE Journal

Biochemistry

7.6K 次观看.  MedImmune. Peptide adsorption to plasticware during traditional tip-based serial dilutions can significantly impact potency determination and confound the understanding of structure-activity relationships used for lead identification and lead optimization phases of drug discovery. Here methods for automated acoustic non-contact serial dilution of peptide samples are described. 

视频: Automated Acoustic Dispensing for the Serial Dilution of Peptide Agonists in Potency Determination Assays

Preparation and Delivery of Protein Microcrystals in Lipidic Cubic Phase for Serial Femtosecond Crystallography

Membrane proteins (MPs) are essential components of cellular membranes and primary drug targets. Rational drug design relies on precise structural information, typically obtained by crystallography; however MPs are difficult to crystallize. Recent progress in MP structural determination has benefited greatly from the development of lipidic cubic phase (LCP) crystallization methods, which typically yield well-diffracting, but often small crystals that suffer from radiation damage during traditional crystallographic data collection at synchrotron sources. The development of new-generation X-ray free-electron laser (XFEL) sources that produce extremely bright femtosecond pulses has enabled room temperature data collection from microcrystals with no or negligible radiation damage. Our recent efforts in combining LCP technology with serial femtosecond crystallography (LCP-SFX) have resulted in high-resolution structures of several human G protein-coupled receptors, which represent a notoriously difficult target for structure determination. In the LCP-SFX technique, LCP is recruited as a matrix for both growth and delivery of MP microcrystals to the intersection of the injector stream with an XFEL beam for crystallographic data collection. It has been demonstrated that LCP-SFX can substantially improve the diffraction resolution when only sub-10 &#181;m crystals are available, or when the use of smaller crystals at room temperature can overcome various problems associated with larger cryocooled crystals, such as accumulation of defects, high mosaicity and cryocooling artifacts. Future advancements in X-ray sources and detector technologies should make serial crystallography highly attractive and practicable for implementation not only at XFELs, but also at more accessible synchrotron beamlines. Here we present detailed visual protocols for the preparation, characterization and delivery of microcrystals in LCP for serial crystallography experiments. These protocols include methods for conducting crystallization experiments in syringes, detecting and characterizing the crystal samples, optimizing crystal density, loading microcrystal laden LCP into the injector device and delivering the sample to the beam for data collection.

We describe procedures for the preparation and delivery of membrane protein microcrystals in lipidic cubic phase for serial crystallography at X-ray free-electron lasers and synchrotron sources. These protocols can also be applied for incorporation and delivery of soluble protein microcrystals, leading to substantially reduced sample consumption compared to liquid injection.

制备及脂质立方相反应蛋白微晶交付串行飞秒晶体

Membrane proteins (MPs) are essential components of cellular membranes and primary drug targets. Rational drug design relies on precise structural ...

科研

生物化学

Automated Protocols for Macromolecular Crystallization at the MRC Laboratory of Molecular Biology

When high quality crystals are obtained that diffract X-rays, the crystal structure may be solved at near atomic resolution. The conditions to crystallize proteins, DNAs, RNAs, and their complexes can however not be predicted. Employing a broad variety of conditions is a way to increase the yield of quality diffraction crystals. Two fully automated systems have been developed at the MRC Laboratory of Molecular Biology (Cambridge, England, MRC-LMB) that facilitate crystallization screening against 1,920 initial conditions by vapor diffusion in nanoliter droplets. Semi-automated protocols have also been developed to optimize conditions by changing the concentrations of reagents, the pH, or by introducing additives that potentially enhance properties of the resulting crystals. All the corresponding protocols will be described in detail and briefly discussed. Taken together, they enable convenient and highly efficient macromolecular crystallization in a multi-user facility, while giving the users control over key parameters of their experiments.

Automated systems and protocols for the routine preparation of a large number of screens and nanoliter crystallization droplets for vapor diffusion experiments are described and discussed.

分子生物学实验室中大分子结晶的自动化协议

When high quality crystals are obtained that diffract X-rays, the crystal structure may be solved at near atomic resolution. The conditions to crystallize ...

Easy Manipulation of Architectures in Protein-based Hydrogels for Cell Culture Applications

Hydrogels are recognized as promising materials for cell culture applications due to their ability to provide highly hydrated cell environments. The field of 3D templates is rising due to the potential resemblance of those materials to the natural extracellular matrix. Protein-based hydrogels are particularly promising because they can easily be functionalized and can achieve defined structures with adjustable physicochemical properties. However, the production of macroporous 3D templates for cell culture applications using natural materials is often limited by their weaker mechanical properties compared to those of synthetic materials. Here, different methods were evaluated to produce macroporous bovine serum albumin (BSA)-based hydrogel systems, with adjustable pore sizes in the range of 10 to 70 &#181;m in radius. Furthermore, a method to generate channels in this protein-based material that are several hundred microns long was established. The different methods to produce pores, as well as the influence of pore size on material properties such as swelling ratio, pH, temperature stability, and enzymatic degradation behavior, were analyzed. Pore sizes were investigated in the native, swollen state of the hydrogels using confocal laser scanning microscopy. The feasibility for cell culture applications was evaluated using a cell-adhesive RGD peptide modification of the protein system and two model cell lines: human breast cancer cells (A549) and adenocarcinomic human alveolar basal epithelial cells (MCF7).

Different methods to manipulate three-dimensional architecture in protein-based hydrogels are evaluated here with respect to material properties. The macroporous networks are functionalized with a cell-adhesive peptide, and their feasibility in cell culture is evaluated using two different model cell lines.

便于操作的体系结构基于蛋白质的凝胶中为单元格文化应用程序

Hydrogels are recognized as promising materials for cell culture applications due to their ability to provide highly hydrated cell environments. The field ...

Analysis of Histone Antibody Specificity with Peptide Microarrays

Post-translational modifications (PTMs) on histone proteins are widely studied for their roles in regulating chromatin structure and gene expression. The mass production and distribution of antibodies specific to histone PTMs has greatly facilitated research on these marks. As histone PTM antibodies are key reagents for many chromatin biochemistry applications, rigorous analysis of antibody specificity is necessary for accurate data interpretation and continued progress in the field. This protocol describes an integrated pipeline for the design, fabrication and use of peptide microarrays for profiling the specificity of histone antibodies. The design and analysis aspects of this procedure are facilitated by ArrayNinja, an open-source and interactive software package we recently developed to streamline the customization of microarray print formats. This pipeline has been used to screen a large number of commercially available and widely used histone PTM antibodies, and data generated from these experiments are freely available through an online and expanding Histone Antibody Specificity Database. Beyond histones, the general methodology described herein can be applied broadly to the analysis of PTM-specific antibodies.

This manuscript describes methods for applying peptide microarray technology to specificity profiling of antibodies that recognize histones and their post-translational modifications.

用肽微阵列分析组蛋白抗体特异性

Post-translational modifications (PTMs) on histone proteins are widely studied for their roles in regulating chromatin structure and gene expression. The ...

Semi-automated Biopanning of Bacterial Display Libraries for Peptide Affinity Reagent Discovery and Analysis of Resulting Isolates

Biopanning bacterial display libraries is a proven technique for peptide affinity reagent discovery for recognition of both biotic and abiotic targets. Peptide affinity reagents can be used for similar applications to antibodies, including sensing and therapeutics, but are more robust and able to perform in more extreme environments. Specific enrichment of peptide capture agents to a protein target of interest is enhanced using semi-automated sorting methods which improve binding and wash steps and therefore decrease the occurrence of false positive binders. A semi-automated sorting method is described herein for use with a commercial automated magnetic-activated cell sorting device with an unconstrained bacterial display sorting library expressing random 15-mer peptides. With slight modifications, these methods are extendable to other automated devices, other sorting libraries, and other organisms. A primary goal of this work is to provide a comprehensive methodology and expound the thought process applied in analyzing and minimizing the resulting pool of candidates. These techniques include analysis of on-cell binding using fluorescence-activated cell sorting (FACS), to assess affinity and specificity during sorting and in comparing individual candidates, and the analysis of peptide sequences to identify trends and consensus sequences for understanding and potentially improving the affinity to and specificity for the target of interest.

Biopanning bacterial display libraries is a proven technique for discovery of peptide affinity reagents, a robust alternative to antibodies. The semi-automated sorting method herein has streamlined biopanning to decrease the occurrence of false positives. Here we illustrate the thought process and techniques applied in evaluating candidates and minimizing downstream analysis.

细菌显示文库的半自动化筛选肽亲和试剂的发现与分离结果分析

Biopanning bacterial display libraries is a proven technique for peptide affinity reagent discovery for recognition of both biotic and abiotic targets. ...

Determination of the Glycogen Content in Cyanobacteria

Cyanobacteria accumulate glycogen as a major intracellular carbon and energy storage&#160;during photosynthesis. Recent developments in research have highlighted complex mechanisms of glycogen metabolism, including the diel cycle of biosynthesis and catabolism, redox regulation, and the involvement of non-coding RNA. At the same time, efforts are being made to redirect carbon from glycogen to desirable products in genetically engineered cyanobacteria to enhance product yields. Several methods are used to determine the glycogen contents in cyanobacteria, with variable accuracies and technical complexities. Here, we provide a detailed protocol for the reliable determination of the glycogen content in cyanobacteria that can be performed in a standard life science laboratory. The protocol entails the selective precipitation of glycogen from the cell lysate and the enzymatic depolymerization of glycogen to generate glucose monomers, which are detected by a glucose oxidase-peroxidase (GOD-POD) enzyme coupled assay. The method has been applied to Synechocystis sp. PCC 6803 and Synechococcus sp. PCC 7002, two model cyanobacterial species that are widely used in metabolic engineering. Moreover, the method successfully showed differences in the glycogen contents between the wildtype and mutants defective in regulatory elements or glycogen biosynthetic genes.

Here, we present a reliable and easy assay to measure the glycogen content in cyanobacterial cells. The procedure entails precipitation, selectable depolymerization, and the detection of glucose residues. This method is suitable for both wildtype and genetically engineered strains and can facilitate the metabolic engineering of cyanobacteria.

测定蓝细菌中的糖原含量

Cyanobacteria accumulate glycogen as a major intracellular carbon and energy storage&#160;during photosynthesis. Recent developments in research have ...

Personalized Peptide Arrays for Detection of HLA Alloantibodies in Organ Transplantation

In organ transplantation, the function and longevity of the graft critically rely on the success of controlling immunological rejection reactivity against human leukocyte antigens (HLA). Histocompatibility guidelines are based on laboratory tests of anti-HLA immunity, which presents either as pre-existing or de novo generated HLA antibodies that constitute a major transplantation barrier. Current tests are built on a single-antigen beads (SAB) platform using a fixed set of ~100 preselected recombinant HLA antigens to probe transplant sera. However, in humans there exist a far greater variety of HLA types, with no two individuals other than identical twins who can share the same combination of HLA sequences. While advanced technologies for HLA typing and direct sequencing can precisely capture any mismatches in DNA sequence between a donor's and recipient's HLA, the SAB assay, due to its limited variety in sequence representation, is unable to precisely detect alloantibodies specifically against the donor HLA mismatches. We sought to develop a complementary method using a different technology to detect and characterize anti-donor HLA antibodies on a personalized basis. The screening tool is a custom peptide array of donor HLA-derived sequences for probing post-transplant sera of the organ recipient to assess the risk for antibody-mediated rejection. On a single array for one donor-recipient pair, up to 600 unique peptides are made based on the donor's HLA protein sequences, each peptide carrying at least one mismatched residue in a 15-amino acid sequence. In our pilot experiments to compare antigen patterns for pre- and post-transplant sera on these arrays, we were able to detect anti-HLA signals with the resolution that also allowed us to pinpoint the immune epitopes involved. These personalized antigen arrays allow high-resolution detection of donor-specific HLA epitopes in organ transplantation.

Mismatches in human leukocyte antigen (HLA) sequences between organ donor and recipient pairs are the major cause of antibody-mediated rejection in organ transplantation. Here we present the use of custom antigen arrays that are based on individual donors' HLA sequences to probe anti-donor HLA alloantibodies in organ recipients.

个性化肽阵列在器官移植 HLA 抗体检测中的研究

In organ transplantation, the function and longevity of the graft critically rely on the success of controlling immunological rejection reactivity against ...

Growing Protein Crystals with Distinct Dimensions Using Automated Crystallization Coupled with In Situ Dynamic Light Scattering

The automated crystallization device is a patented technique1 especially developed for monitoring protein crystallization experiments with the aim to precisely maneuver the nucleation and crystal growth towards desired sizes of protein crystals. The controlled crystallization is based on sample investigation with in situ Dynamic Light Scattering (DLS) while all visual changes in the droplet are monitored online with the help of a microscope coupled to a CCD camera, thus enabling a full investigation of the protein droplet during all stages of crystallization. The use of in situ DLS measurements throughout the entire experiment allows a precise identification of the highly supersaturated protein solution transitioning to a new phase &#8211; the formation of crystal nuclei. By identifying the protein nucleation stage, the crystallization can be optimized from large protein crystals to the production of protein microcrystals. The experimental protocol shows an interactive crystallization approach based on precise automated steps such as precipitant addition, water evaporation for inducing high supersaturation, and sample dilution for slowing induced homogeneous nucleation or reversing phase transitions.

Growing Protein Crystals with Distinct Dimensions Using Automated Crystallization Coupled with In Situ Dynamic Light Scattering

Here we present a protocol for controlled production of protein microcrystals. The process uses an automated device allowing controlled manipulation of several crystallization parameters. The protein crystallization is carried-out by controlled and automated addition of crystallization solutions while monitoring and investigating the radius distribution of particles in the crystallization droplet.

采用自动结晶耦合原位动态光散射的不同尺寸生长蛋白晶体

The automated crystallization device is a patented technique1 especially developed for monitoring protein crystallization experiments with the aim to ...

Enabling Real-Time Compensation in Fast Photochemical Oxidations of Proteins for the Determination of Protein Topography Changes

Fast photochemical oxidation of proteins (FPOP) is a mass spectrometry-based structural biology technique that probes the solvent-accessible surface area of proteins. This technique relies on the reaction of amino acid side chains with hydroxyl radicals freely diffusing in solution. FPOP generates these radicals in situ by laser photolysis of hydrogen peroxide, creating a burst of hydroxyl radicals that is depleted on the order of a microsecond. When these hydroxyl radicals react with a solvent-accessible amino acid side chain, the reaction products exhibit a mass shift that can be measured and quantified by mass spectrometry. Since the rate of reaction of an amino acid depends in part on the average solvent accessible surface of that amino acid, measured changes in the amount of oxidation of a given region of a protein can be directly correlated to changes in the solvent accessibility of that region between different conformations (e.g., ligand-bound versus ligand-free, monomer vs. aggregate, etc.) FPOP has been applied in a number of problems in biology, including protein-protein interactions, protein conformational changes, and protein-ligand binding. As the available concentration of hydroxyl radicals varies based on many experimental conditions in the FPOP experiment, it is important to monitor the effective radical dose to which the protein analyte is exposed. This monitoring is efficiently achieved by incorporating an inline dosimeter to measure the signal from the FPOP reaction, with laser fluence adjusted in real-time to achieve the desired amount of oxidation. With this compensation, changes in protein topography reflecting conformational changes, ligand-binding surfaces, and/or protein-protein interaction interfaces can be determined in heterogeneous samples using relatively low sample amounts.

Fast photochemical oxidation of proteins is an emerging technique for the structural characterization of proteins. Different solvent additives and ligands have varied hydroxyl radical scavenging properties. To compare the protein structure in different conditions, real-time compensation of hydroxyl radicals generated in the reaction is required to normalize reaction conditions.

实现蛋白质快速光化学氧化的实时补偿，用于确定蛋白质地形变化

Fast photochemical oxidation of proteins (FPOP) is a mass spectrometry-based structural biology technique that probes the solvent-accessible surface area ...

Optimizing the Growth of Endothiapepsin Crystals for Serial Crystallography Experiments

Here, a protocol is presented to facilitate the creation of large volumes (&#62; 100 &#181;L) of micro-crystalline slurries suitable for serial crystallography experiments at both synchrotrons and XFELs. The method is based upon an understanding of the protein crystal phase diagram, and how that knowledge can be utilized. The method is divided into three stages: (1) optimizing crystal morphology, (2) transitioning to batch, and (3) scaling. Stage 1 involves finding well diffracting, single crystals, hopefully but not necessarily, presenting in a cube-like morphology. In Stage 2, the Stage 1 condition is optimized by crystal growth time. This strategy can transform crystals grown by vapor diffusion to batch. Once crystal growth can occur within approximately 24 h, a morphogram of the protein and precipitant mixture can be plotted and used as the basis for a scaling strategy (Stage 3). When crystals can be grown in batch, scaling can be attempted, and the crystal size and concentration optimized as the volume is increased. Endothiapepsin has been used as a demonstration protein for this protocol. Some of the decisions presented are specific to endothiapepsin. However, it is hoped that the way they have been applied will inspire a way of thinking about this procedure that others can adapt to their own projects.

The aim of this article is to give the viewer a solid understanding of how to transform their small-volume, vapor-diffusion protocol, for growing large, single protein crystals, into a large-volume batch micro-crystallization method for serial crystallography.