该议定书是从上一个出版物11通过的。t.z.、d. Č和 J. 由捷克共和国教育、青年和体育部在国家可持续发展方案 (NPU 一) 的支持下, 授予编号 LO1415。J. Č也得到了 GA CR 的支持, 18-24397S 号。捷克的系统生物学 C4SYS 研究基础设施 (项目没有 LM2015055) 支持获得仪器和其他设施的机会。m.a.s. 得到了俄罗斯科学基金会的资助 [14-14-00904]。

1. 蓝藻栽培

<ol><li>培养Synechocystis细胞在锥形烧瓶或光生物反应器10,19在缓冲 BG11 中等20保持 pH < 10 (例如, 使用17毫米 HEPES10)。 注: 标准栽培条件要求受控温度 (通常为30摄氏度, 最佳温度为35°c)21, 光照 (通常是强度高达800µmol [光子]/[m2·s])21的白色光, 和 CO2供应 (在400毫升平板光生物反应器, 增长饱和 CO2集中是 1700 ppm)21。</li>
	<li>要确定培养密度, 测量的文化光学密度 spectrophotometrically 在730毫微米 (OD730) 使用一个试管以 1 cm 的光路径. 或者, 使用 hemocytometer 或自动单元格在显微镜下计数细胞计数器。</li>
</ol>2. 样品准备
<ol><li>低辐照度下工作, 防止蛋白降解。</li>
	<li>收获 3 x 1 毫升的文化悬浮到安全锁管。使用 triplicates 来估计测量中的技术误差。在无菌条件下进行培养取样, 在层流罩中收获细胞, 并遵循适当的工作和安全做法。</li>
	<li>在实验室温度下将细胞在 1.5万 x g处离心成5分钟. 注意适当平衡的离心机转子。离心后, 丢弃上清液。一定不要打扰球团。</li>
	<li>把样品放在冰箱里。要长期贮存, 请将样品保持在摄氏-80 摄氏度。这是防止蛋白退化的必要措施。对于短期储存,-20 °c 是足够的。</li>
	<li>冷冻干燥样品过夜。在适当的冷冻干燥过程中, 将冷冻干燥冷凝器的温度保持在-60 摄氏度以下, 冷冻干燥室的压力约为1帕。</li>
	<li>完成冷冻干燥循环后, 应尽快关闭管, 以防止吸收的空气中的水。</li>
</ol>3. 细胞均匀化和色素提取
<ol><li>将四片玻璃珠 (直径2毫米) 添加到每个取样管上, 然后关闭管。 注: 当用于均匀化的安全锁管的盖子太薄时, 可以在匀质过程中断裂;因此, 本协议只推荐有强盖的安全锁管。</li>
	<li>融汇样品与玻璃珠十五年代在一个均匀机在实验室温度。 注: 在安全锁管的整个内表面均有适当的匀质试样。</li>
	<li>添加1毫升的 PBS 缓冲器 (pH 7.4), 预换热器到4摄氏度, 以提取藻胆蛋白的样品。 注意: 从这个步骤上, 保持样品在冰上, 以防止降解的提取蛋白。这是至关重要的。</li>
	<li>在实验室温度下, 将样品与 PBS 混合5秒。 注: 混合后, 样品呈绿色。</li>
	<li>混合后, 将样品放在冰上60分钟, 用盖子盖住冰浴, 以防颜料降解。</li>
	<li>经过60分钟的蛋白萃取, 离心机的样品在 1.5万 x克4 摄氏度为5分钟. 注意适当平衡离心机转子。 注: 离心后, 上清有青色蓝色。</li>
</ol>4. 蛋白量化
<ol><li>在分光光度测量之前, 用 PBS 缓冲器将光谱仪校准为空白。</li>
	<li>校准分光光度计后, 将 PBS 从一个分光光度计试管, 用提取的藻胆蛋白代替所丢弃的缓冲器, 将上清液吸管移除。</li>
	<li>用 0.5 nm 的狭缝宽度量化蛋白浓度 spectrophotometrically。<ol>
<li>分别测量藻和复方阿司匹林中 phycocyanobilins 在 615 nm (615) 和 652 nm (652) 的 PBS 缓冲器上的吸光度, 并测量在 720 nm (720) 的细胞碎片的吸收量。</li>
		<li>根据班尼特和 Bogorad12的方程式 (1) 和 (2) 计算藻和复方阿司匹林的浓度: <img alt="Equation 1" src="/files/ftp_upload/58076/58076eq1.jpg"> <img alt="Equation 2" src="/files/ftp_upload/58076/58076eq2.jpg"> 注:615、652和720应符合分光光度计的线性吸收范围。如有必要, 用 PBS 缓冲器稀释样品。</li>
	</ol>
</li>
	<li>重新计算原始样品中的颜料浓度。样品中的颜料浓度与方程 (1) 和 (2) 的结果直接对应, 当1毫升的培养基和1毫升的萃取缓冲器用于分析时。如果使用不同数量的蓝藻样品和/或 PBS 缓冲器, 最后的颜料浓度应根据等式 (3) 计算: <img alt="Equation 3" src="/files/ftp_upload/58076/58076eq3.jpg">(3) 注: 在每细胞干重的正则蛋白含量的情况下, 应根据方程式 (4)<img alt="Equation 4" src="/files/ftp_upload/58076/58076eq4.jpg"> </img> (4) 计算最终颜料浓度。</li>
</ol>5. 测定细胞干重 (可选)
<ol><li>在分析天平上重三个空的安全锁管。 注意: 安全锁管干燥是至关重要的。如果将管子贮存在潮湿的环境中, 将管子在冷冻干燥机中干燥几个小时, 然后再称量。轻轻地操纵管子, 只用无粉手套的手, 以避免材料和研究员的手指之间的任何接触。保持所有的持有者和离心机的清洁, 以避免任何材料转移到管。</li>
	<li>样品 1 x 15 毫升的文化悬浮到15毫升圆锥管。</li>
	<li>离心机的培养悬浮在15毫升圆锥管在 4000 x g在实验室温度为10分钟. 平衡离心机转子与三额外15毫升圆锥管, 每个包含15毫升的水。离心后, 丢弃12毫升的上清。</li>
	<li>并用重悬颗粒在剩余的上清和转移 3 x 1 毫升混合物到三1.5 毫升安全锁管子与吸管。如果剩余的颗粒留在15毫升圆锥管, 添加1.5 毫升的去离子水到锥形管, 涡流或摇动管并用重悬余下的颗粒, 并转移 3 x 0.5 毫升的混合物到三1.5 毫升安全锁管 (已经 containing 3 x 1 毫升的颗粒) 与吸管。 注: 将球团除以三1.5 毫升的安全锁管, 可以估计干重测量的任何技术误差。</li>
	<li>在实验室温度为5分钟, 将1.5 毫升安全锁管中的细胞离心成 1.5万 x克. 平衡离心机转子与另外1.5 毫升安全锁管, 包含1毫升水。离心后, 丢弃上清液。</li>
	<li>把样品放在冰箱里。长期贮存, 样品保持在摄氏-80 摄氏度;为短期存贮,-20 °c 是充足的。</li>
	<li>冷冻干燥样品过夜。为适当的冷冻干燥过程, 保持冷冻干燥冷凝器的温度低于-90 °c 和在冷冻干燥室的压力约 1 hPa。</li>
	<li>冷冻干燥后, 关闭管, 并权衡样品的分析平衡。 注: 干重值可用于每细胞干重蛋白含量的规范化。</li>
</ol>

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C., 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=Stable+isolation+of+phycocyanin+from+Spirulina+platensis+associated+with+high-pressure+extraction+process.">Stable isolation of phycocyanin from Spirulina platensis associated with high-pressure extraction process.</a> International Journal of Molecular Sciences. 14 (1), 1778-1787 (2013).</li><li>Touloupakis, E., Cicchi, B., Torzillo, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+bioenergetic+assessment+of+photosynthetic+growth+of+Synechocystis+sp.+PCC+6803+in+continuous+cultures.">A bioenergetic assessment of photosynthetic growth of Synechocystis sp. PCC 6803 in continuous cultures.</a> Biotechnology for Biofuels. 8 (1), 133 (2015).</li><li>Touloupakis, E., Cicchi, B., Benavides, A. M. S., Torzillo, G. <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+high+pH+on+growth+of+Synechocystis+sp.+PCC+6803+cultures+and+their+contamination+by+golden+algae+(Poterioochromonas+sp.).">Effect of high pH on growth of Synechocystis sp. PCC 6803 cultures and their contamination by golden algae (Poterioochromonas sp.).</a> Applied Microbiology and Biotechnology. 100 (3), 1333-1341 (2016).</li><li>Ishii, A., Hihara, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+AbrB-Like+Transcriptional+Regulator,+Sll0822,+Is+Essential+for+the+Activation+of+Nitrogen-Regulated+Genes+in+Synechocystis+sp.+PCC+6803.">An AbrB-Like Transcriptional Regulator, Sll0822, Is Essential for the Activation of Nitrogen-Regulated Genes in Synechocystis sp. PCC 6803.</a> Plant Physiology. 148 (1), 660-670 (2008).</li></ol>

作者没有什么可透露的。

该协议描述了一种简单、快速、可重现的方法, 用于量化蓝藻Synechocystis中的蛋白内容。比较了细胞均匀化、蛋白质提取、藻和复方阿司匹林量化的几种方法, 最后的协议代表了每一个过程的最优步骤的组合。作为代表性数据, 藻胆蛋白在Synechocystis细胞中的含量随着光照强度的增加而定量化。尽管分析需要类似的时间和实验室设备作为一些以前发布的方法12,13,14,15,16, 这个协议的优点是不将叶绿素a释放到萃取缓冲器中, 从而可以估计出蛋白测量精度高。
蛋白分析所需的细胞悬浮量可能随培养密度而变化。1毫升的样品容量被优选了为文化与 OD730 1.5, 细胞密度大约 2 x 107个细胞或毫升, 或者为文化以大约20毫克/L 的蛋白内容。在稀释的文化情况下, 收获一个更大的文化容量。另一方面, 在密集的文化情况下, 收获一个较低的文化体积-在高密度的文化, 有一个风险, 藻胆蛋白部分留在细胞后提取。同样, 干重测定所需的细胞悬浮量也随培养密度而变化。15毫升的取样体积被优化的文化与 OD730 1.5 或与细胞密度约 2 x 107细胞/毫升。与低密度文化或以更低的容量, 测量误差可能增加。相反, 更大的文化容量或密集的文化提供更好的方法决议。
该协议的关键步骤是细胞均匀化和蛋白提取, 应提供高产量和高特异性。通过冷冻干燥样品, 将干细胞颗粒均匀化成玻璃微珠, 并在 PBS 缓冲器中进行蛋白萃取, 达到了同时最大蛋白质保存的提取物的最高产量和纯度 (图 1)。由于叶绿素a对粗蛋白提取物的少量污染可能导致蛋白含量过高, 因此有必要通过添加 PBS 缓冲器来避免任何叶绿素的提取。超声波用于细胞破坏时, 在粗提取物中检测到叶绿素a 。如图 1所示, 重复超声波循环, 提取物中叶绿素a的数量增加。另一方面, 在重复超声波循环后, 提取的蛋白质 (由 280 nm 的吸收量检测) 也增加了。因此, 超声波的使用 (结合液氮中的冻融循环) 似乎是一种适用于总蛋白提取的方法 (游离和膜结合蛋白从细胞中释放出来);但是, 不建议用蛋白分光光度法进行量化。钟等。建议使用1% 硫酸链霉素, 以消除膜碎片与叶绿素a16.在这里, 硫酸链霉素的使用似乎没有必要, 因为它没有导致减少叶绿素a在蛋白质提取物 (图 7)。尽管120s 的单个超声波周期没有有效地中断单元格 (方法 B 和 C,图 1), 其他方法 (如图 1中所示的方法 F 或图 7B中所示的方法) 确认了超声波的早期发现是一种有效的细胞破坏方法22,28。另一方面, 简单的冻融循环, 以前也报告为藻胆蛋白萃取22,28的有效方法, 提供了最低的产量在这里描述的测试 (方法 E,图1).用锆珠进行的细胞均匀化 (萃取缓冲器内, 2 x 六十年代) 测试的效率低于冻干细胞颗粒的 15-均匀性 (方法 D,图 1)。在提取缓冲区内的均匀化过程前面描述了一个较长的均匀化时间 (10 分钟)29。我们没有测试均匀化超过2分钟, 因为样品在每个均匀化周期中显著升温。在文献中还描述了其他细胞破坏方法, 包括使用法国新闻16或磨床13,15,30;然而, 这项研究并没有对这些方法进行测试。
蛋白提取是在十五年代 (图 2) 在 PBS 缓冲区进行的。缓冲 pH 值为 7.4, 先前报告为蛋白萃取22的最佳选择。氯化钠或氯化钾的加入没有提高蛋白萃取效率 (图 3), 这与以前的观察22相抵触。然而, 如图 3所示, 本议定书所使用的磷酸盐缓冲盐水溶液中含有154毫米氯化钠 (除5.6 毫米 Na2HPO4和1毫米), 这不允许我们建立无 NaCl 的 PBS 提取缓冲器作为控制。我们还比较了 PBS 缓冲器和醋酸钠缓冲液16提取效率。磷酸磷酸钠和磷酸钾在 PBS 缓冲器中的结合提供了更高的藻胆蛋白产量 (图 3)。这一发现与 Hemlata 和al的先前发现相符。22。
蛋白萃取时间优化为 1 h. 长时间萃取并没有导致显著的蛋白降解或萃取改善 (图 4), 这与 Lawrenz 和al的以前的工作相对应。28. 提取时间短于1小时, 未进行测试。同样, 提取超过4小时没有测试, 因为它以前发现, 提取的藻胆蛋白在磷酸盐缓冲稳定至少48小时28。
通过对已知蛋白质的商业标准中两种藻胆蛋白的含量的重新计算, 比较了文献中所描述的几种分光光度藻和复方阿司匹林量化方程。浓度 (图 6)。用班尼特和 Bogorad12的方程式实现了两种颜料的最佳重建。这一结果并不特定于磷酸盐提取缓冲器, 因为这样的缓冲器以前使用过12、13、15。它不是具体的颜料标准, 因为615,618和620 (用于藻估计的波长在早先工作) 在藻标准是1% 和区别之间650和652 (以前使用的波长为复方阿司匹林估计12,13,14,16) 在复方阿司匹林标准是3%。同样, Synechocystis蛋白提取物中的615和620之间的差异只有2%。这种在吸收光谱上的微小差异不会导致最终的颜料变异达 36% (图 6)。因此, 个别方程式之间的差异是相当连接到变化的蛋白消光系数的特定有机体12,13,14,15,16,17. 有趣的是, 钟et方程。提供了最低的藻重建 (图 6), 虽然作者也使用Synechocystis为他们的实验16。
建议在 280-720 nm 之间测量蛋白萃取物的连续光谱, 而不是单波长的测定, 以估计萃取物中叶绿素a的存在, 这可能会干扰复方阿司匹林和藻吸收。此外, 通过测量 280 nm (280) 的吸光度, 两个藻的纯度 (615/280或620/280)21,22和复方阿司匹林 (652/280)24内提取物可估计 (0.7: 食品级, 3.9: 反应级, > 4.0: 分析级)21。
作为代表性数据, 藻和复方阿司匹林在Synechocystis中的浓度在光照强度的增加下确定。在 turbidostat 栽培制度的11、21中保持恒定水平的培养密度是直接比较试验的必要条件。藻和复方阿司匹林浓度为 2.4%-10.2% 和 0.6%-1.7% 的细胞干重, 分别与先前报告的值11,31,32相似。此外, 每个单元格的蛋白内容与以前报告的11、33相似。随着光照的增加, 藻胆蛋白含量下降。有趣的是, 即使在1100年µmol (光子)/(m2·s) 的最高光强度下, 藻胆蛋白也没有完全漂白。
由于该议定书只要求标准设备, 而且相对较快, 因此可以很容易地在任何藻胆蛋白进行例行分析的实验室采用。

fPhycobiliproteins 是水溶性色素蛋白复合物, 代表原核蓝藻 (蓝藻) 和几种真核生物群中的光收获触角的主要成分 (Glaucophyta、红藻门和Cryptophyta)1。它们主要发生在称为 phycobilisomes 的超分子复合体上, 通常附着在基质侧的光合膜表面, 除Cryptophyta外, 藻胆蛋白在囊体腔2。迄今已确定了四种类型的藻胆蛋白: 核心复方阿司匹林和外围藻、藻红蛋白和 phycoerythrocyanin1。phycobilisomes 作为主要的光收获配合物, 是藻类和蓝藻群体培养生产力的重要因素之一。结果表明, phycobilisomes 截断可提高强光下3的生物量积累。另一方面, 在适度或低辐照度下, 天线截断导致生长速率和生物量积累减少3,4。藻胆蛋白在化妆品行业中作为食品着色剂、药品和食品添加剂, 并作为荧光探针应用于流式细胞术、荧光免疫和荧光显微镜5。
本协议着重于蓝藻Synechocystis模型中藻胆蛋白的定量测定。蓝藻是最早的氧光合自养;他们已经形成了地球的生物圈超过24亿年6。它们在氮、碳、氧和其他元素的全球生物地球化学循环中起着至关重要的作用。在蓝藻中, 单细胞菌株Synechocystis获得了独特的地位, 因为它是第一个蓝藻与整个基因组序列7,8, 它是自然转化的外源 DNA9, 并它执行稳定和相对地快速的成长10,11。在Synechocystis, 核心天线组件, 复方阿司匹林, 是与整体膜蛋白, 并附上藻位于囊体膜外围。
在该协议中比较了蛋白提取和定量的几种方法。最后提取程序成功地应用于Synechocystis, 以及其他蓝藻菌株, 包括Cyanothece sp, Synechococcuselongatus,螺旋藻sp,节旋藻sp和念珠菌, 也成功地应用于红藻类紫球藻藻。因此, 本协议中开发的方法可以被认为是蛋白提取的通用方法。尽管一些经过测试的提取方法导致了总蛋白产量的提高, 这里描述的萃取过程提供了最高的蛋白产量连同叶绿素的最低含量的残留在蛋白提取物。降低叶绿素a的含量是正确藻和复方阿司匹林分光光度定量的关键。
不同藻类和蓝藻物种的蛋白吸收光谱在12、13、14、15、16、17 、甚至在18种单一蓝藻属的几种菌株中。因此, 用于测定Synechocystis中藻和复方阿司匹林的特定波长和吸收系数一般不适用于其他菌株。此外, Synechocystis不包含藻红蛋白和 phycoerythrocyanin, 可以在其他藻类和蓝藻中发现。为了确定非Synechocystis菌株中的藻胆蛋白, 建议分别对各菌株的分光光度方程进行评价。
虽然该议定书包含两个较长的步骤 (夜间冷冻干燥的细胞颗粒和1小时蛋白质提取), 总的劳动时间为藻胆蛋白量化不超过2小时。

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		<tr height="21">
			<td height="21" style="height:21px;width:407px;">Synechocystis sp. PCC 6803</td>
			<td style="width:417px;">Institut Pasteur, Paris, France</td>
			<td style="width:137px;">6803</td>
			<td style="width:627px;">Cyanobacterium strain</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Roti-CELL PBS</td>
			<td>Carl Roth GmbH + Co. KG, Karlsruhe, Germany</td>
			<td>9143.1</td>
			<td>Phosphate-Buffered Saline (PBS) solution, pH 7.4</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Eppendorf safe-lock tubes&#160;</td>
			<td style="width:417px;">Eppendorf, Hamburk, Germany</td>
			<td>30120086</td>
			<td>Safe-lock tubes 1.5 ml</td>
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		<tr height="21">
			<td height="21" style="height:21px;">VWR 80-Place Storage System</td>
			<td style="width:417px;">VWR International, Radnor, Pennsylvania, USA</td>
			<td>30128-282</td>
			<td>Holder for safe-lock tubes&#160;</td>
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		<tr height="21">
			<td height="21" style="height:21px;">RAININ 100 &#181;l -1000 &#181;l&#160;</td>
			<td>Mettler-Toledo, Columbus, Ohio, USA</td>
			<td>17014382</td>
			<td>Pipette</td>
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		<tr height="21">
			<td height="21" style="height:21px;">GP-LTS-A-1000&#181;L-/F-768/8</td>
			<td>Mettler-Toledo, Columbus, Ohio, USA</td>
			<td>30389272</td>
			<td>Pipette tips</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Rotina 420R</td>
			<td>Hettich, Kirchlengern, Germany</td>
			<td>4701</td>
			<td>Refrigerated centrifuge for 1.5 ml safe-lock tubes and 15 ml conical centrifuge tubes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LCexv 4010</td>
			<td>Liebherr, Bulle, Switzerland</td>
			<td>9005382197172</td>
			<td>Refrigerator and freezer -20&#160;&#176;C</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:407px;">Revco ExF -86&#176;C Upright Ultra-Low Temperature Freezer</td>
			<td style="width:417px;">Thermo Fisher Scientific, Waltham, Massachusetts, USA</td>
			<td style="width:137px;">EXF24086V&#160;</td>
			<td>Freezer -80&#160;&#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CoolSafe</td>
			<td>LaboGene, Liller&#248;d, Denmark</td>
			<td>7.001.000.615</td>
			<td>Freeze dryer&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">UV-2600</td>
			<td>Shimadzu, Kyoto, Japan</td>
			<td>UV-2600</td>
			<td>Spectrophotometer&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hellma absorption cuvettes, semi Micro</td>
			<td>Sigma-Aldrich, St. Louis, Missouri, USA</td>
			<td>Z600288&#160;</td>
			<td>VIS/UV-VIS semi-micro cuvettes 0.75-1.5 ml, spectral range 200-2500 nm&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Silamat S6</td>
			<td>Ivoclar Vivadent, Schaan, Liechtenstein</td>
			<td>602286WU</td>
			<td>Homogenizer&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Solid-glass beads</td>
			<td>Sigma-Aldrich, St. Louis, Missouri, USA</td>
			<td>Z273627</td>
			<td>Glass bead of the diameter 2&#160;mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CPA225D-0CE</td>
			<td>Sartorius AG, G&#246;ttingen, Germany</td>
			<td>SECURA225D-1OBR</td>
			<td>Analytical balances</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">C-Phycocyanin from Spirulina sp.&#160;</td>
			<td>Sigma-Aldrich, St. Louis, Missouri, USA</td>
			<td>P2172</td>
			<td>Phycocyanin standard</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Allophycocyanin</td>
			<td>Sigma-Aldrich, St. Louis, Missouri, USA</td>
			<td>A7472</td>
			<td>Allophycocyanin standard</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:407px;">Bicinchoninic Acid Kit&#160;</td>
			<td style="width:417px;">Sigma-Aldrich, St. Louis, Missouri, USA</td>
			<td style="width:137px;">BCA1, B9643</td>
			<td>Complete kit for total proteins determination</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:407px;">AlgaeTron&#160;</td>
			<td style="width:417px;">Photon System Instruments Ltd., Dr&#225;sov, Czech Republic</td>
			<td style="width:137px;">AG 130-ECO&#160;</td>
			<td>Cultivation chamber for E. flasks, with controllable light and atmosphere</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:407px;">Photobioreactor</td>
			<td style="width:417px;">Photon System Instruments Ltd., Dr&#225;sov, Czech Republic</td>
			<td style="width:137px;">FMT-150</td>
			<td>Cultivation equipment for cyanobacteria and algae with completely controllable environment</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cellometer&#160;</td>
			<td style="width:417px;">Nexcelom Bioscience, Lawrence, Massachusetts, USA</td>
			<td style="width:137px;">Auto M10</td>
			<td>Cell counter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:407px;">Corning 15 mL centrifuge tubes</td>
			<td style="width:417px;">Sigma-Aldrich, St. Louis, Missouri, USA</td>
			<td style="width:137px;">CLS430791&#160;</td>
			<td>15 ml Centrifuge tube for dry weigth sampling</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:407px;">Herasafe KS</td>
			<td style="width:417px;">Thermo Fisher Scientific, Waltham, Massachusetts, USA</td>
			<td style="width:137px;">51024579</td>
			<td>Laminar flow hood</td>
		</tr>
	</tbody>
</table>

spectrophotometric determination of phycobiliprotein content in cyanobacterium synechocystis

这是一个简单的协议, 以定量测定蛋白的内容在模型蓝藻Synechocystis。藻胆蛋白是 phycobilisomes 中最重要的成分, 是蓝藻中主要的光捕获触角和几种藻类群。Synechocystis的 phycobilisomes 包含两个藻胆蛋白: 藻和复方阿司匹林。该协议描述了一种简单、高效、可靠的方法, 用于定量测定该模型蓝藻中的藻和复方阿司匹林。比较了几种蛋白提取方法和分光光度法。本协议中所述的萃取过程也成功地应用于其他蓝藻菌株, 如Cyanothece 、 Synechococcuselongatus、螺旋藻、节旋藻sp、和念珠菌, 以及红藻类紫球藻藻。然而, 不同分类群的特定藻胆蛋白的消光系数可能不同, 因此建议分别验证每一个单株的分光光度法。该议定书需要很少的时间, 可以在任何标准生命科学实验室进行, 因为它只需要标准设备。

对于最初的方法测试, Synechocystis被培养了作为批培养在锥形烧瓶在 BG11 耕种媒介20 (补充与17毫米 HEPES) 在25°c, 在一个温暖的白色光之下强度的50µmol (光子)/(m2·s) 和与 1% CO2在培养的大气。在养殖过程中, 将这些培养物取样到安全锁管和离心 (实验室温度为5分钟 1.5万克), 将清液丢弃, 并将样品储存在-80 摄氏度, 冷冻干燥作为制备本协议的个别步骤或存储在-20 °c 或-80 °c 进行比较提取和 quantifications 分析。
经过测试的萃取过程包括超声波的细胞分裂, PBS 缓冲中的氧化锆珠均匀化, 以及冻融。图 1总结了不同提取方法的结果。本协议中描述的方法提供了最高的藻胆蛋白产量, 以及提取缓冲区中的最低叶绿素a污染。当超声波用于细胞中断时, 在萃取缓冲器中检测到叶绿素a 。在冰上的超声波浴中, 冷冻和解冻样品的重复循环导致了萃取缓冲器中蛋白质产量的升高。但同时, 萃取缓冲液中叶绿素a浓度增加 (图 1 ), 排除了适当的蛋白含量量化。
最佳均匀化时间为十五年代. 在较短 (5 秒) 和更长 (二十年代) 期间的均匀化提供了稍低的蛋白收益率, 如图 2所示。同直径为0.5 毫米、1毫米或4毫米的玻璃珠相比, 用直径为2毫米的玻璃珠均匀化的细胞, 其直径为0.5 毫米的氧化锆珠, 或与海砂颗粒相比较。PBS 提取缓冲器被测试为藻胆蛋白提取的最佳缓冲器 (图 3)。增加100毫米氯化钠或150毫米氯化钾到 PBS 缓冲或对水没有提高蛋白提取效率, 并且同样去使用20毫米乙酸钠缓冲为提取 (图 3)。最佳蛋白提取时间为60分钟, 因为在更多时间 (最多240分钟) 后, 提取缓冲区中的蛋白浓度没有显著变化 (图 4)。
我们还测试了蛋白测量的分光光度计线性范围 (图 5), 并比较了蛋白量化的几个方程式, 同时使用了藻和复方阿司匹林标准 (图 6)。本协议中使用的分光光度计显示了 0.1-3.0 之间的线性吸光度范围 (图 5)。因为蛋白质提取物的光谱有它的最大吸收大约620毫微米 (620), 并且在652毫微米, 吸收 (652) 仅 33% 620 (图 7),分光光度计线性为652 (最大复方阿司匹林吸光度) 仅测试了652的1.16。班尼特和 Bogorad12的方程组 (1) 和 (2) 为所有已测试的方程式中的藻和复方阿司匹林标准提供了最佳的重建 (图 6)。硫酸链霉素是用于消除含有叶绿素a的膜碎片的前几项研究中所使用的, 它不需要提取 (图 7)。
在代表实验中, Synechocystis是在一个 turbidostat 的光生物反应器25中培养的, 细胞在一个定义的光学密度范围内维持在指数生长阶段 (测量在 680 nm, OD680) 由一个被控制的稀释由一个新鲜的耕种媒介 (BG11 媒介缓冲与17毫米 HEPES)11,26。文化耕种了在32°c 和输入空气包含了 0.5% CO2。文化被照亮了以红色光 (λ最大: 633 毫微米, λ1/2:20 毫微米) 强度的 25-1100 µmol (光子)/(m2·s), 连同蓝色光 (λ最大: 445 毫微米, λ1/2:20 毫微米) 强度25µmol (光子)/(m2·s)。培养悬浮液的光学密度由光生物反应器基测量, OD680的范围设置为 0.52-0.58 (2 x 107到 4 x 107细胞/毫升)。在每一个特定的光照强度下培养至少24小时, 以提供足够的时间进行驯化。在达到生长稳定后, 这些培养物被取样以干重 (根据本协议的步骤 2.1-2.8)、细胞计数、藻和复方阿司匹林含量。图 8显示了在增加光强度下蛋白评价的结果。在最高辐照度下, Synechocystis将蛋白含量从12% 的细胞干重 (505 个成品/细胞) 降低到3% 的细胞干重 (280 个成品/细胞)。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58076/58076fig1.jpg"> 图 1: 比较了本协议中描述的方法的提取效率和几种参考方法.测定粗蛋白提取物中藻 (黑条) 和复方阿司匹林 (白条) 的浓度, 在 PBS 缓冲器中提取60分钟. 方法 A 在本协议中描述。方法 B 被修改如下: 收获后, 细胞离心 (1.5万 x g在实验室温度为5分钟), 并保存在-20 °c 过夜。冷冻样品为120s 的微气泡为4摄氏度, 藻胆蛋白在 PBS 缓冲器中提取60分钟. 方法 C 从方法 B 中进行了改进, 将收获细胞的颗粒储存在-80 °c 30 分钟, 冷冻干燥细胞颗粒, 并将 PBS 缓冲器添加到干颗粒中。方法 D 由中、铝改性。16: 冷冻干燥后 (与方法 C 相同), 0.25 毫升的钠醋酸盐与1% 毫升的硫酸链霉素缓冲 (pH 5.5) 被添加到干细胞颗粒中, 连同 0.25 mL 锆珠 (直径为0.5 毫米), 样品均匀地两次。六十年代在均质机上。在均匀化后, 在样品中加入0.5 毫升的钠-醋酸盐, 1% 硫酸链霉素缓冲液 (pH 5.5), vortexed, 蛋白质在冰上提取30分钟. 方法 E 由 PBS 缓冲器中的单一冻融循环和蛋白质提取24小时在4摄氏度。方法 F (嵌入图) 包括六循环冻结细胞 (最初, 冷冻干燥和悬浮在 PBS 缓冲) 在液氮, 其次是超声波冰。由于大量叶绿素在粗蛋白提取物中存在, 藻胆蛋白在方法 F 中没有量化。所描述的值表示来自三技术复制的平均值, 误差线表示标准偏差。根据本协议的方程式 (1) 和 (2) 计算颜料浓度。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58076/58076fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58076/58076fig2.jpg"> 图 2: 均匀化时间对蛋白提取效率的影响.Synechocystis细胞的冻干颗粒 (根据本协议的步骤 3.1-3.5) 在 5 s、十五年代和二十年代的均质机上被玻璃珠打断。所描述的值表示来自三技术复制的平均值, 误差线表示标准偏差。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58076/58076fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58076/58076fig3.jpg"> 图 3: 蛋白提取效率在各种提取缓冲器.藻胆蛋白在 pbs 缓冲器中提取, 在 pbs 缓冲器中加入100毫米氯化钠或150毫米氯化钾, 在去离子水中有100毫米氯化钠或150毫米氯化钾, 根据 Hemlata 和 Fareha22, 在20毫米醋酸钠中, 根据钟et/c4>。16. 根据本议定书步骤4.1 至 4.3, 提取的情况为4摄氏度。所描述的值表示来自三技术复制的平均值, 误差线表示标准偏差。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58076/58076fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/58076/58076fig4.jpg"> 图 4: 蛋白在萃取缓冲器中的稳定性及时.在提取过程中, 按照本协议步骤 4.1-4.3 所述执行, PBS 缓冲器中的蛋白提取物 (pH 7.4) 存储在4摄氏度, 长达4小时, 没有显著的藻 (圆圈) 和复方阿司匹林 (平方) 退化。虚线表示用最小二乘方法计算的时间点的线性拟合。所描述的值表示来自三技术复制的平均值, 误差线表示标准偏差。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58076/58076fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/58076/58076fig5.jpg"> 图 5: 藻和复方阿司匹林浓度的代表性测量Synechocystis。致密的培养悬浮液 (藻: 456 µg/毫升, 复方阿司匹林: 106 µg/毫升) 逐渐稀释到19µg/毫升浓度为藻和复方阿司匹林。所描述的值表示来自三技术复制的平均值, 误差线表示标准偏差。虚线表示用最小二乘法计算的浓度点的线性拟合。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58076/58076fig5large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/58076/58076fig6.jpg"> 图 6: 颜料标准中藻和复方阿司匹林浓度的估算.根据藻和 Bogorad12、Lüder 和 spectrophotometrically 的方程, 对颜料标准中的复方阿司匹林含量进行了测定. 13, Sampath-威利和 Neefus15, 埃文斯14, 和钟等 al。16. 标准中的蛋白质含量 (藻胆蛋白测定的必要) 是用二辛可宁酸、碳酸钠、酒石酸钠和碳酸氢钠在 0.1 N 氢氧化钠中的溶液 (最后 pH 为 11.25) 来量化的, 在反应与 4% (w/v) 硫酸铜铜 (II)27, 使用牛血清白蛋白作为蛋白质标准。班尼特和 Bogorad 的方程式提供了两种颜料的最高重建: 96% 的藻标准和99% 的复方阿司匹林标准。所描述的值表示来自三技术复制的平均值, 误差线表示标准偏差。虚线突出显示了100% 点。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58076/58076fig6large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/58076/58076fig7.jpg"> 图 7: 硫酸链霉素对 PBS 缓冲液中粗蛋白提取物吸收谱的影响.(A) 本议定书步骤 4.1-4.3 中所述的提取程序或 (B) 使用超声波, 如图 1的图例所述的方法 F 在 pbs 缓冲区 (圆圈) 和 pbs 缓冲区中进行, 辅以1%硫酸链霉素 (正方形)。蛋白量化是根据该协议的步骤 5.1-5.4 执行的。所描述的值表示来自三技术复制的平均值, 误差线表示标准偏差。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58076/58076fig7large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/58076/58076fig8.jpg"> 图 8: 藻 (圆) 和复方阿司匹林 (正方形) 的浓度在Synechocystis栽培下的红橙光强度为 25-1100 µmol (光子)/(m2·s)。Synechocystis培养在光生物反应器在32°c, 与 0.5% CO2在输入空气, 在 BG11 耕种媒介补充17毫米 HEPES 在 turbidostat 政权根据 Zavrel等。11. 按照本议定书的规定, 对蛋白浓度进行了测量, 按照本议定书2节的规定, 对每个细胞干重的最终蛋白内容进行了规范化。虚线表示测量点的功率拟合, 用最小二乘方法计算。所描述的值表示来自三技术复制的平均值, 误差线表示标准偏差。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58076/58076fig8large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Spectrophotometric Determination of Phycobiliprotein Content in Cyanobacterium Synechocystis at JoVE.com

Spectrophotometric Determination of Phycobiliprotein Content in Cyanobacterium Synechocystis

在这里, 我们提出了一个协议, 以定量地确定蛋白的内容在蓝藻Synechocystis使用分光光度法。提取过程也成功地应用于其他蓝藻和藻类菌株;然而, 由于颜料吸收谱的变化, 有必要分别测试每种菌株的分光光度方程。

分光光度法测定蓝藻Synechocystis中的蛋白含量

This is a simple protocol for the quantitative determination of phycobiliprotein content in the model cyanobacterium Synechocystis. Phycobiliproteins are ...

spectrophotometric-determination-phycobiliprotein-content

Nanyang Technological University

Research

JoVE Journal

Biochemistry

Spectrophotometric Determination of Phycobiliprotein Content in Cyanobacterium Synechocystis

15.1K 次观看.  Czech Academy of Sciences. 在这里, 我们提出了一个协议, 以定量地确定蛋白的内容在蓝藻Synechocystis使用分光光度法。提取过程也成功地应用于其他蓝藻和藻类菌株;然而, 由于颜料吸收谱的变化, 有必要分别测试每种菌株的分光光度方程。

视频: Spectrophotometric Determination of Phycobiliprotein Content in Cyanobacterium Synechocystis

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

As with small molecule drug discovery, screening for peptide agonists requires the serial dilution of peptides to produce concentration-response curves. Screening peptides affords an additional layer of complexity as conventional tip-based sample handling methods expose peptides to a large surface area of plasticware, providing an increased opportunity for peptide loss via adsorption. Preventing excessive exposure to plasticware reduces peptide loss via adherence to plastics and thus minimizes inaccuracies in potency prediction, and we have previously described the benefits of non-contact acoustic dispensing for in vitro high-throughput screening of peptide agonists1. Here we discuss a fully integrated automation solution for non-contact acoustic preparation of peptide serial dilutions in microtiter plates utilizing the example of screening for peptide agonists at the mouse glucagon-like peptide-1 receptor (GLP-1R). Our methods allow for high-throughput cell-based assays to screen for agonists and are easily scalable to support increased sample throughput, or to allow for increased numbers of assay plate copies (e.g., for a panel of more target cell lines).

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. ...

科研

生物化学

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 ...

High Throughput, Absolute Determination of the Content of a Selected Protein at Tissue Levels Using Quantitative Dot Blot Analysis (QDB)

Lacking a convenient, quantitative, high throughput immunoblot method for absolute determination of the content of a specific protein at cellular and tissue level significantly hampers the progress in proteomic research. Results derived from currently available immunoblot techniques are also relative, preventing any efforts to combine independent studies with a large-scale analysis of protein samples. In this study, we demonstrate the process of quantitative dot blot analysis (QDB) to achieve absolute quantification in a high throughput format. Using a commercially available protein standard, we are able to determine the absolute content of capping actin protein, gelsolin-like (CAPG) in protein samples prepared from three different mouse tissues (kidney, spleen, and prostate) together with a detailed explanation of the experimental details. We propose the QDB analysis as a convenient, quantitative, high throughput immunoblot method of absolute quantification of individual proteins at the cellular and tissue level. This method will substantially aid biomarker validation and pathway verification in various areas of biological and biomedical research.

High Throughput, Absolute Determination of the Content of a Selected Protein at Tissue Levels Using Quantitative Dot Blot Analysis QDB

Here we demonstrate a detailed process of quantitative dot blot analysis (QDB) by determining the absolute content of a targeted protein, capping actin protein, gelsolin-like (CAPG), in three different mouse tissues. We demonstrate a high throughput, convenient, quantitative immunoblot technique for biomarker validation at the cellular and tissue level.

高通量, 用定量斑点杂交分析法绝对测定组织层中选定蛋白质的含量 (QDB)

Lacking a convenient, quantitative, high throughput immunoblot method for absolute determination of the content of a specific protein at cellular and ...

A Colorimetric Method for Measuring Iron Content in Plants

Iron, one of the most important micronutrients in living organisms, is involved in basic processes, such as respiration and photosynthesis. Iron content is rather low in all organisms, amounting in plants to about 0.009% of dry weight. To date, one of the most accurate methods for measuring iron concentration in plant tissues is flame absorption atomic spectroscopy. However, this approach is time-consuming and expensive and requires specific equipment not commonly found in plant laboratories. Therefore, a simpler, yet accurate method that can be routinely used is needed. The colorimetric Prussian Blue method is regularly used for qualitative iron staining in animal and plant histological sections. In this study, we adapted the Prussian Blue method for quantitative measurements of iron in tobacco leaves. We validated the accuracy of this method using both atomic spectroscopy and Prussian Blue staining to measure iron content in the same samples and found a linear regression (R2 = 0.988) between the two procedures. We conclude that the Prussian Blue method for quantitative iron measurement in plant tissues is precise, simple, and inexpensive. However, the linear regression presented here may not be appropriate for other plant species, due to potential interactions between the sample and the reagent. Establishment of a regression curve is thus needed for different plant species.

We present a simple and reliable protocol for measuring iron content in plant tissues using the colorimetric Prussian Blue method.

测定植物铁含量的比色法

Iron, one of the most important micronutrients in living organisms, is involved in basic processes, such as respiration and photosynthesis. Iron content ...

Using High Content Imaging to Quantify Target Engagement in Adherent Cells

Quantitating the interaction of small molecules with their intended protein target is critical for drug development, target validation and chemical probe validation. Methods that measure this phenomenon without modification of the protein target or small molecule are particularly valuable though technically challenging. The cellular thermal shift assay (CETSA) is one technique to monitor target engagement in living cells. Here, we describe an adaptation of the original CETSA protocol, which allows for high throughput measurements while retaining subcellular localization at the single cell level. We believe this protocol offers important advances to the application of CETSA for in-depth characterization of compound-target interaction, especially in heterogeneous populations of cells.

Measurements of drug target engagement are central to effective drug development and chemical probe validation. Here, we detail a protocol for measuring drug-target engagement using high content imaging in a microplate-compatible adaption of the cellular thermal shift assay (CETSA).

利用高含量成像对增强细胞中的目标参与进行量化

Quantitating the interaction of small molecules with their intended protein target is critical for drug development, target validation and chemical probe ...

Enrichment of Native Lipoprotein Particles with microRNA and Subsequent Determination of Their Absolute/Relative microRNA Content and Their Cellular Transfer Rate

Lipoprotein particles are predominately transporters of lipids and cholesterol in the bloodstream. Furthermore, they contain small amounts of strands of noncoding microRNA (miRNA). In general, miRNA alters the protein expression profile due to interactions with messenger-RNA (mRNA). Thus, knowledge of the relative and absolute miRNA content of lipoprotein particles is essential to estimate the biological effect of cellular particle uptake. Here, a quantitative real-time polymerase chain reaction (qPCR)-based protocol is presented to determine the absolute miRNA content of lipoprotein particles&#8212;exemplified shown for native and miRNA-enriched lipoprotein particles. The relative miRNA content is quantified using multiwell microfluidic array cards. Furthermore, this protocol allows scientists to estimate the cellular miRNA and, thus, the lipoprotein particle uptake rate. A significant increase of the cellular miRNA level is observable when using high-density lipoprotein (HDL) particles artificially loaded with miRNA, whereas incubation with native HDL particles yields no significant effect due to their rather low miRNA content. In contrast, the cellular uptake of low-density lipoprotein (LDL) particles&#8212;neither with native miRNA nor artificially loaded with it&#8212;did not alter the cellular miRNA level.

Here, a quantitative real-time polymerase chain reaction-based protocol is presented for the determination of the native micro-RNA content (absolute/relative) of lipoprotein particles. In addition, a method for increasing the micro-RNA level, as well as a method for determining the cellular uptake rate of lipoprotein particles, is demonstrated.

微 Rna 对原生脂蛋白颗粒的富集及其与绝对相对微 Rna 含量及细胞转移速率的测定

Lipoprotein particles are predominately transporters of lipids and cholesterol in the bloodstream. Furthermore, they contain small amounts of strands of ...

Spectrophotometric Screening for Potential Inhibitors of Cytosolic Glutathione S-Transferases

Glutathione S-transferases (GSTs) are metabolic enzymes responsible for the elimination of endogenous or exogenous electrophilic compounds by glutathione (GSH) conjugation. In addition, GSTs are regulators of mitogen-activated protein kinases (MAPKs) involved in apoptotic pathways. Overexpression of GSTs is correlated with decreased therapeutic efficacy among patients undergoing chemotherapy with electrophilic alkylating agents. Using GST inhibitors may be a potential solution to reverse this tendency and augment treatment potency. Achieving this goal requires the discovery of such compounds, with an accurate, quick, and easy enzyme assay. A spectrophotometric protocol using 1-chloro-2,4-dinitrobenzene (CDNB) as the substrate is the most employed method in the literature. However, already described GST inhibition experiments do not provide a protocol detailing each stage of an optimal inhibition assay, such as the measurement of the Michaelis-Menten constant (Km) for CDNB or indication of the employed enzyme concentration, crucial parameters to assess the inhibition potency of a tested compound. Hence, with this protocol, we describe each step of an optimized spectrophotometric GST enzyme assay, to screen libraries of potential inhibitors. We explain the calculation of both the half-maximal inhibitory concentration (IC50) and the constant of inhibition (Ki)&#8212;two characteristics used to measure the potency of an enzyme inhibitor. The method described can be implemented using a pool of GSTs extracted from cells or pure recombinant human GSTs, namely GST alpha 1 (GSTA1), GST mu 1 (GSTM1) or GST pi 1 (GSTP1). However, this protocol cannot be applied to GST theta 1 (GSTT1), as CDNB is not a substrate for this isoform. This method was used to test the inhibition potency of curcumin using GSTs from equine liver. Curcumin is a molecule exhibiting anti-cancer properties and showed affinity towards GST isoforms after in silico docking predictions. We demonstrated that curcumin is a potent competitive GST inhibitor, with an IC50 of 31.6 &#177; 3.6 &#181;M and a Ki of 23.2 &#177; 3.2 &#181;M. Curcumin has potential to be combined with electrophilic chemotherapy medication to improve its efficacy.

Glutathione S-transferases (GSTs) are detoxification enzymes involved in the metabolism of numerous chemotherapeutic drugs. Overexpression of GSTs is correlated with cancer chemotherapy resistance. One way to counter this phenotype is to use inhibitors. This protocol describes a method using a spectrophotometric assay to screen for potential GST inhibitors.

细胞性谷胱甘肽S-转移酶潜在抑制剂的分光光度筛选

Glutathione S-transferases (GSTs) are metabolic enzymes responsible for the elimination of endogenous or exogenous electrophilic compounds by glutathione ...

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 ...

Rapid Determination of Antibody-Antigen Affinity by Mass Photometry

Measurements of the specificity and affinity of antigen-antibody interactions are critically important for medical and research applications. In this protocol, we describe the implementation of a new single-molecule technique, mass photometry (MP), for this purpose. MP is a label- and immobilization-free technique that detects and quantifies molecular masses and populations of antibodies and antigen-antibody complexes on a single-molecule level. MP analyzes the antigen-antibody sample within minutes, allowing for the precise determination of the binding affinity and simultaneously providing information on the stoichiometry and the oligomeric state of the proteins. This is a simple and straightforward technique that requires only picomole quantities of protein and no expensive consumables. The same procedure can be used to study protein-protein binding for proteins with a molecular mass larger than 50 kDa. For multivalent protein interactions, the affinities of multiple binding sites can be obtained in a single measurement. However, the single-molecule mode of measurement and the lack of labeling imposes some experimental limitations. This method gives the best results when applied to measurements of sub-micromolar interaction affinities, antigens with a molecular mass of 20 kDa or larger, and relatively pure protein samples. We also describe the procedure for performing the required fitting and calculation steps using basic data analysis software.

We describe a single-molecule approach to antigen-antibody affinity measurements using mass photometry (MP). The MP-based protocol is fast, accurate, uses a very small amount of material, and does not require protein modification.

通过质量光度法快速测定抗体抗原亲和力

Measurements of the specificity and affinity of antigen-antibody interactions are critically important for medical and research applications. In this ...

Crystallization and Structural Determination of an Enzyme:Substrate Complex by Serial Crystallography in a Versatile Microfluidic Chip

The preparation of well diffracting crystals and their handling before their X-ray analysis are two critical steps of biocrystallographic studies. We describe a versatile microfluidic chip that enables the production of crystals by the efficient method of counter-diffusion. The convection-free environment provided by the microfluidic channels is ideal for crystal growth and useful to diffuse a substrate into the active site of the crystalline enzyme. Here we applied this approach to the CCA-adding enzyme of the psychrophilic bacterium Planococcus halocryophilus in the presented example. After crystallization and substrate diffusion/soaking, the crystal structure of the enzyme:substrate complex was determined at room temperature by serial crystallography and the analysis of multiple crystals directly inside the chip. The whole procedure preserves the genuine diffraction properties of the samples because it requires no crystal handling.

A versatile microfluidic device is described that enables the crystallization of an enzyme using the counter-diffusion method, the introduction of a substrate in the crystals by soaking, and the 3D structure determination of the enzyme:substrate complex by a serial analysis of crystals inside the chip at room temperature.

酶的结晶和结构确定:多功能微流体芯片中串行晶体学的基板复合物

The preparation of well diffracting crystals and their handling before their X-ray analysis are two critical steps of biocrystallographic studies. We ...