LMB 结晶设施得到医学研究委员会 (英国) 的支持。我们感谢 LMB 的成员们的支持: 奥尔加佩里希奇 (PNAC), 托尼沃恩, Fusinita 范窝耳鼻喉科和帕特爱德华兹 (结构研究), 史蒂夫 Scotcher 和其他成员的机械车间, 尼尔格兰特和 Jo (视觉教具), 保罗哈特和汤姆普拉特 (它)。我们也要感谢史蒂夫艾略特 (Tecan, 英国), 米切尔和希瑟 Ringrose (汉密尔顿机器人学, 英国), 保罗解冻, 罗伯特刘易斯和 Joby 詹金斯 (Labtech, 英国), 保罗里尔登 (Swissci AG, 瑞士), 乔治斯蒂芬斯和唐纳德 Ogg (Alphabiotech,英国), 尼尔威廉斯 (马肯玛士, 英国) 和格雷厄姆哈里斯 (克利夫兰代办处) 为技术帮助。

1. 两个全自动系统 (初步筛选)
<ol><li>
系统 1: 用筛包 (LMB 板) 填充的 72 96-井结晶板的制备
	<ol>
<li>在执行该过程之前, 请确保系统的机器人已初始化, 并且其控制软件处于打开状态。在主程序启动前30分钟, 打开管冷却托架的冷水机。</li>
		<li>在平板封口机的机动 SBS 载体上放置一个测试板。运行印版封口机, 检查胶片是否正确应用。重复此测试两次, 以验证板封口机是否已就绪。</li>
		<li>然后, 在液体处理器的主容器中加入20升去离子水。从小容器 (20% 乙醇漂洗液) 中断开耦合插入, 并将刀片连接到主容器。</li>
		<li>在喷墨打印机触摸屏上输入适当的屏幕名称 (表 1)。然后, 打开并关闭旋转木马的门, 以触发旋转木马。当第一个栈呈现自己时, 打开门。</li>
		<li>充分装载第一个堆栈与22结晶板, 每个与专栏1朝向外面。以相同的方式完全加载下两个堆栈。</li>
		<li>将剩余的6盘装入第四叠的最高位置。然后, 关闭旋转木马的门。</li>
		<li>验证冷却器显示14° c。轻轻地反复翻转所选的筛包1分钟。然后, 打开液体处理程序的前面板。</li>
		<li>按照标准的96井布局 (A1、A2、等) 打开管道并将其放置在冷却托架中。将每根管子放置在托架上时, 将其盖子放在同一96井布局中的托盘上。</li>
		<li>交叉检查管子的位置, 确保所有的管子都是水平的, 并在托架中固定。然后, 关闭前面板。</li>
		<li>在液体处理程序软件的 "启动" 窗口中, 选择 "运行维护" 并打开冲洗程序。运行程序从系统中冲洗20% 乙醇, 并清洗液体配药技巧的外部。</li>
		<li>刷新完成后, 单击 "取消" 返回 "启动"。选择 "运行现有进程", 然后单击 "开始选择"。打开 "工具包配药" 程序。在配置屏幕中为 "实例" 填写 "18", 然后运行该程序。</li>
		<li>监测系统, 因为前四板的标签, 填充和密封。</li>
		<li>一旦所有的72板块都准备就绪, 并放回传送带中, 从主容器到小容器的耦合插入。运行 "启动刷新" 程序 (请参见步骤 1.1.10)。</li>
		<li>关闭冷却器并丢弃空的屏蔽套件管。小心地从传送带上取下72准备好的盘子。丢弃不正确填充或密封不良的板。在10° c 的时候储存现成的盘子。</li>
	</ol>
</li>
	<li>
系统 2: 在 20 LMB 板中从单一样品中设置结晶液滴 (100 nl 蛋白 + 100 nl 条件)
	<ol>
<li>首先, 确保系统的机器人是开着的, nanoliter 分配器是用其软件打开的, 而液体处理程序的方法管理器是打开的。在主程序启动前15分钟打开微冷却托架。</li>
		<li>在平板封口机的机动 SBS 载体上放置一个测试板。运行该板封口机, 并验证该板是否正确密封。测试板封口机三次。</li>
		<li>然后, 运行 nanoliter 分配器在测试板中设置 100-nL 测试液滴。在显微镜下检查水滴的设置是否正确。关闭 nanoliter 分配器软件, 并从甲板上卸下带座架。</li>
		<li>接下来, 在定制设计的平板夹中插入 (请参见代表性结果: 在 LMB 中开发的结晶装置) LMB 板, 其条件中的挥发性试剂的相对数量最高 (表 1)。从板上取下粘合膜。</li>
		<li>打开液体处理程序前面板, 并将未密封的板放在甲板上, 在第一滑动托架的背面 (滑动载体可以拉出以便于访问)。</li>
		<li>用 SBS 铝盖盖板。通过施加柔和的压力到盖子的对面角落, 解决盖子对托架的后方左角落。</li>
		<li>开封, 加载到滑动载体, 并以同样的方式覆盖其余的19板块, 工作从最不稳定的条件。一旦微冷却的载体是在4° c, 如指示由绿灯, 去除它的盖子。</li>
		<li>切断含有 (至少) 440 µL 蛋白质样本 (表 2) 的微的盖子。确保样品没有泡沫以上的半月板, 因为这将干扰液体检测系统。将管放置在微冷却托架的1位置。</li>
		<li>在平板移动的适配器托架前面的液体处理器甲板上放置一个 PCR 板。然后, 关闭前面板。</li>
		<li>在装载甲板之后, 确保 nanoliter 分配器甲板是清楚的条带持有人块, 并且载体在 50-µL 尖叠的后面是清楚的铝 SBS 盖子。</li>
		<li>在液体处理程序的 "方法管理" 界面中, 选择 "设置板"。监视两个系统的初始化并填充运行参数。遵循方法管理界面 (提示、数字和提示管理系统帮助准备) 的指导原则。</li>
		<li>检查所有必需的组件是否已就绪, 然后启动该进程。</li>
		<li>当 nanoliter 分配器将第一个板中的水滴设置为系统时, 对其进行监控, 然后将板封口机封上。</li>
		<li>一旦所有20板都已准备好结晶液滴, 并自动返回到滑动载体, 打开前面板, 轻轻地卸下板。在储存结晶之前, 请检查板材是否正确密封。</li>
		<li>用20% 乙醇溶液清洗 SBS 盖, 然后将其堆放在液体处理程序的左手边以存放。丢弃 PCR 板和微。</li>
		<li>关闭微冷却托架, 然后擦去冷凝。在冷却器表面上留一条纸巾, 以吸收进一步的冷凝。然后, 更换托架盖并关闭前面板。</li>
	</ol>
</li>
</ol>2. 条件的优化
<ol><li>
基于注射器的液体处理: 产生两个线性梯度的浓度到储层的结晶板 (4 角法)。
	<ol>
<li>首先, 确保液体处理程序打开并初始化其软件。</li>
		<li>在 "渐变" 选项卡上: 打开所需的程序, 选择储层中的结晶板类型和最终体积 (表 3)。"最大铅球卷" 的高级设置应在使用含有 [异丙醇] > 10% v/v 和 [MPD] > 20% v/五的解决方案时从6000降至 3000
</li>
		<li>准备注射器在每个注射器中放置一个活塞 (指向末端), 然后将注射器的背部插入机器人头部下面的指定凹槽中。顺时针旋转注射器锁定它的位置 (程序将只启动与所有所需的注射器正确连接)。</li>
		<li>准备水槽拆卸不锈钢框架并插入槽。左边的4位置对应于4角 A, B, C, d 切换到 "设置" 选项卡, 显示每个注射器所需的解决方案量 (在表 3中, 0.5 mL 死卷被添加到显示的卷中)。将角部溶液倒入各自的槽中, 然后将车架放回甲板上 (框架保持位置, 2 小磁铁位于甲板前部)。另一种继续执行此步骤的方法是将解决方案倒入槽中, 当它们已经放置在甲板上时.
</li>
		<li>将结晶板放在机动 SBS 载体上。</li>
		<li>点击 ' 吸气 ', 等待这一步完成 (当活塞停止时, 他们的方式)。</li>
		<li>切换到 "运行" 选项卡并运行程序。</li>
		<li>程序完成后, 返回 "设置" 选项卡, 然后单击 "删除": 系统从剩余的解决方案中清除注射器, 然后将活塞向上抬起。可以请求 "清除", 而不是 "删除"; 这将使活塞处于底部位置, 准备用相同的注射器吸入更多的溶液.
</li>
		<li>通过逆时针扭转注射器。</li>
		<li>在适当的纸盒中丢弃注射器和水槽 (或用去离子水冲洗, 然后用20% 伏/v 乙醇溶液清洗)。</li>
		<li>密封板, 并把它放在微搅拌机3分钟 1000 rpm, 或10分钟, 当高度粘性的解决方案是混合。该板准备在 nanoliter 分配器上设置结晶液滴。</li>
	</ol>
</li>
	<li>
加性筛选协议 1 (在96井结晶板上设置结晶液滴, 加装预填充)
	<ol>
<li>首先, 准备的条件与初始浓度的试剂增加了 10% (分钟 vol. 15 毫升时, 从一个容器到添加剂屏幕上的液体处理程序) 的条件。</li>
		<li>确保液体处理程序准备好操作。打开程序 ' 添加屏幕到添加剂 ' 为一个单一的板块 (进入容积在水库: ' 72 µL ')。提示、数字和提示管理系统 (需要 12 x 1000 µL 提示) 帮助确保机器人可以根据选择进行操作.
</li>
		<li>从-20 ° c 的恒温箱中取出添加剂屏幕, 并立即取下其铝封 (使用盘架), 然后将板放在液体处理器的甲板上。程序根据纵向布局进行操作 (该板放置在位于托架左前方的 A1-corner)也安置装于情况的容器。运行该程序。</li>
		<li>一旦该板块的水库已经填补, 把它放在微振动筛和运行程序。用去离子水和20% 乙醇清洗条件容器以供再利用。</li>
		<li>
设置 nanoliter 分配器上的液滴。首先, 开封的结晶板 (使用板持有人)。然后, 将该板和8井蛋白带放在带夹座的第一位置。控制软件上的 "设置" 选项卡显示了甲板上每个组件的实际位置。根据所需的放置大小 (表 4), 将带蛋白质样品的长条装入每个井。运行程序以准备液滴.
</li>
		<li>程序完成后, 将板从甲板上卸下, 并立即将其密封 (使用板封口机, 3 英寸宽的胶带)。在相应的纸盒中丢弃该条。</li>
		<li>在贮存前评估显微镜下水滴的大小、形状和定心。</li>
	</ol>
</li>
	<li>
添加剂筛选协议 2 (在96井结晶板中设置具有可重新使用的添加剂屏幕的结晶液滴)
	<ol>
<li>首先, 准备加网: 将相应的冷冻96孔细胞培养板在室温下解冻40分钟。然后, 离心的添加剂屏幕在 1000 x g 为2分钟。</li>
		<li>准备条件 (最小 vol. 15 毫升时, 从一个容器转移到添加剂屏幕上的液体处理程序)。</li>
		<li>填装一个96井结晶板的水库以情况。在液体处理程序上, 与协议1步2.2.2 相同, 但在储层中输入 "80 µL"。</li>
		<li>设置 nanoliter 分配器上的液滴, 在甲板上安装3组件 (包含加性屏幕的板, 以及在带座块的第一个位置的晶板和8井蛋白带,表 4)。</li>
		<li>密封的细胞培养板含有的屏幕与铝板, 并把它放回-20 ° c 孵化器。</li>
		<li>在贮存前评估显微镜下水滴的大小、形状和定心。</li>
	</ol>
</li>
</ol>

我们特此声明一个冲突的商业利益, 因为 LifeArc 市场化以下项目:96-和 48-好的湄公河板, 湄公河的悬挂滴印章, 睡眠, Pi, 埃和 LMB 结晶屏幕。

1-存储在板中的初始屏幕的准备和使用
由于在贮存过程中, 在某些管中会出现光沉淀或相分离现象, 所以在将其放入盘子之前, 应先将其混合。当一个屏幕是由两个套件 (2 x 48 管), 第一管的第二个套件是放置在位置 E1 的冷却载体。当一个屏幕由4套件 (4 x 24 管), 第二个套件的第一管放置在位置 C1, 第三个套件的第一管放置在位置 E1 和第四套件的第一管放置在位置 G1。在其冷却托架中插入管时, 将盖子放在标准96井景观布局后的托盘上。由于油井的数量是由制造商在盖子上表示的, 这使得交叉检查, 如果所有的管道已被放置在正确的顺序。这也有助于更换正确的盖子上的管子时, 填补了减少数量的板块。
我们将预填充的钢板储存在10° c 处, 以避免在4° c 处结冰和贮存, 这可能导致条件恶化和密封问题。在密封件的内面上通常没有明显的凝结, 所以板被储存了几个月。这是不正确的 LMB05, LMB06, LMB09 和 LMB10 板, 因为这些包含的条件相对较高的浓度挥发性试剂 (表 1)。密封的内侧有少量的冷凝, 降低了密封效率, 在启封板时会造成井间的交叉污染。为了帮助防止在最初的冷却过程中凝结, 盘子可以首先从传送带转移到一个隔热的野餐冷却器, 这是储存在一个晚上4° c 冷室。非常缓慢的冷却最小化了密封井内温度梯度的发展, 从而减少了整个15的冷凝。此外, 一旦板块被储存在10° c 孵化器, 一个内部定制的 SBS 聚苯乙烯盖放在板上的每个栈顶部 (没有显示)。
我们的整套预填充板可以作为一个大的初始屏幕对一个新的, 水溶性, 蛋白质样本。或者, 可以选择较少的板来满足特定的要求。例如, LMB15 和 LMB19 是专门为膜蛋白样本制定的屏幕26,27, 或 LMB20 是一个用重原子制定的屏幕, 以方便实验性的衍射数据的阶段,28 (参见:<a href="https://www.youtube.com/watch?v=Gpb4SypWnQY&t=11s">睡眠蛋白结晶筛的公式化</a>)。
2. 设置结晶液滴
在使用系统2时, 应首先处理具有大量挥发性试剂的筛分试剂盒。这样可以避免 SBS 盖上的橡胶形成凝结, 从而影响盖子的处理和板的密封。SBS 盖子在板的顶部有一点间隙, 这就是为什么它们最初需要对齐 (参见协议, 步骤 1.2.6)。在 PCR 板的井中的蛋白质死的容量是相对地慷慨的 (0.8 µL, 参见表 2的传奇)。请注意, 当使用 nanoliter 分配器在8井条 (表 4) 中单独用蛋白质时, 也会采用同样慷慨的死卷。更小的死的容量可能工作, 然而有些样品坚持提示, 机器人的定标可能变得轻微地不准确, 房间可能比通常温暖,等。所有这些都导致了慷慨的死卷所涵盖的样本损失, 以巩固这种做法。
最近的发展使实验进一步小型化, 因此, 通过集成相应的技术29,30, 可以大大减少筛选结晶条件所需的样本量..但是, 进一步小型化的某些方面需要仔细考虑, 如水滴31的蒸发和微32的操作。
最后, 在设置结晶液滴 (球形上井) 时, 可以将板材的离心 (2000 rpm, 1 分钟) 整合为常规的最后一步。离心产生的液滴的尺寸和形状越一致, 可能会减少重现性问题33,34。当然, 集中的水滴将会用显微镜来缓解以后的实验评估, 因为在整个板块中, 所需的焦距将是相似的。
3. 4 角法的优点
4角方法最重要的优点是它的简单性, 它使错误最小化, 并方便了简单的自动化协议。例如, 4 角解决方案将始终放置在一个液体处理程序的甲板上, 按照相同的布局。此外, 所有程序都基于解决方案之间的固定比率 (图 3C)。 手动准备4角解决方案是首选的自动化处理的解决方案在高浓度, 可以高度粘性。相对快速和准确的吸入/配药, 然后可能在大多数类型的液体处理, 最低要求的优化液体类。然而, 一些角落的解决方案可能仍然过于粘性的机器人运行的液体系统, 有效地运行。这就是为什么我们选择了一个具有正位移的液体处理程序 (图 3B)。
除了2的浓度线性梯度, 第三个组成部分 (即, 一组缓冲器/添加剂) 可以在一个恒定的浓度在一个方便的方式测试。为此, 一个相对地大容量一组核心的角落解答在适当地更高的集中, 不包括组分要变化, 首先准备。然后, 添加包括此组件的库存解决方案来调整最终的浓度。例如, 50 毫升的一套4角解决方案是准备在10% 以上的浓度比最初。这个核心集合然后被分裂成5更小的子集4。最后, 在每个子集中添加了10% 个不同的缓冲 pH 解的体积。
4. 添加剂屏幕的格式和类型
屏幕通常存储在-20 ° c (图 4) 中, 因为它们不经常使用, 并且含有挥发性/不稳定化合物。在深井区块 (1 毫升) 中储存的冷冻添加剂筛网的使用必须提前计划, 因为所有的添加剂解决方案需要12-24 小时才能完全在室温下解冻。此外, 许多用户共享相同的添加屏幕, 可能导致交叉污染问题。最后, 深井块的高度使它们不适合大多数 nanoliter 分配器。作为绕过这些问题的一种方便的解决方案, 屏幕应该从深井块转移到低剖面板 (图 4)。
从历史上看, 包含多种单一试剂 (单浓度) 的加性屏幕非常受欢迎35,36。然而, 其他类型的添加剂屏幕已经开发, 集成混合的添加剂37或减少数量的单一添加剂发现在不同浓度的38。最后, 一个互补的方法是研究添加剂对样品的影响之前的结晶39,40。
5. 更多的考虑
良好做法:大多数屏幕都含有有害或甚至有毒物质, 因此在协议中必须使用适当的个人保护。同样, 机器人的移动部分可能会导致受伤, 特别是当程序正在运行时 (虽然大多数机器人都有紧急停止按钮/系统), 但在尝试手动干扰时。由于涉及的技术复杂性, 定期检查机器人, 屏幕和程序与以前的特征测试样本是重要的持续高水平的重现性。
吞吐量:作为一个征兆, 在4000到8000之间 LMB 板材年年被生产与系统 1 (和以后使用由用户为初步筛选)。当预期营业额低得多时, 不适合在10° c 下储存大量预填充板, 因为在4-5 月后, 某些条件会开始变质和蒸发。对于中小型实验室, 已经实施了不同的自动化协议方法41。
存储和评估实验:在制备液滴后, 在4或18° c 的室温下, 以严格控制的温度 (+/-0.5 ° c 最大偏差) 将板储存在低振动的货架上。实验用冷光源显微镜进行评估。各种自动化成像系统都是商用的, 但是一个应该仔细考虑所有方面: 扫描一个盘子所需的速度是否足够高吞吐量？除了晶体以外的物体是否会干扰自动对焦？图像的质量是否足以发现非常小的晶体 (特别是在水滴的边缘)？42,43,44
结晶条件比较:在仔细调查了最初获得的晶体的性质之后, 您可以使用<a href="http://fms-3.lmb.internal/screens2/search.html">LMB 屏幕数据库</a>或 C6 <a href="http://c6.csiro.au/">Web 工具</a>45来分析各种情况下的趋势和相似性。

x 射线晶体学被广泛应用于进一步提高我们对原子级生物和疾病机制的理解, 并随后协助对药物发现的合理方法1。为此, 纯化和浓缩 (2-50 毫克/毫升) 大分子样品的蛋白质, DNA, RNA, 其他配体, 及其配合物是试用的倾向, 形成有序的三维晶格通过结晶2,3 ,4。当获得高品质的晶体衍射 X 射线, 晶体结构可以解决在近原子分辨率5,6。关键的是, 不能预测新样品的结晶条件, 而高质量晶体的产量通常很低。一个潜在的原因是, 许多感兴趣的样品具有挑战性的生物化学性质, 这使得他们在相应的时间刻度的结晶 (通常是几天) 不稳定。最后, 该过程由生产样品和样品变型所需的时间合成, 并优化其纯化和结晶7,8。
结晶条件是一种沉淀的溶液, 它可以降低样品的溶解度, 而且条件通常也含有缓冲器和添加剂。数以百计的这种试剂非常适合于改变结晶实验的参数, 因为它们有低的倾向, 干扰样品的完整性 (如蛋白质或核酸的展开)。虽然测试数百万个结晶试剂的组合是不可行的, 测试几个到许多筛选套件-制定与各种策略9,10 -是可能的微型试验和自动化协议。在这个角度, 最适合的技术可能是蒸气扩散与 100-200 nL 液滴坐在一个小井之上包含结晶条件 (25-250 µL), 实施在专业结晶板11,12. 当在上井中设置液滴时, 蛋白质的样品和条件通常以1:1 的比率组合为 200 nL 的总体积。机器人 nanoliter 蛋白质的结晶可以采用替代技术和板块, 如油下批次13和脂立方相14 (最近一个被专门应用于跨膜蛋白水中溶解性极差)。
在2000s 代初开始了<a href="http://www2.mrc-lmb.cam.ac.uk/groups/JYL/WWWrobots/robot.html">LMB 的结晶装置</a>, 并在 2005年15中介绍了我们的自动化协议的早期摘要。介绍了蛋白质结晶的历史介绍, 同时也概述了机器人 nanoliter 方法的优点 (然后是一种新的常规实验方法)。由于大分子结晶实质上是一个随机过程, 很少或没有有用的先验信息, 采用了广泛的 (合适的) 初始条件, 提高了质量衍射晶体的成品率16。此外, 一个大的初始屏幕经常被忽视的优势是大大减少了对样品和晶体的优化需要在许多情况下。当然, 你可能仍然需要继续优化一些初始条件后。通常情况下, 试剂的浓度和 pH 值, 然后系统地调查。更多的试剂也可以引入到优化条件 (s) 进一步改变结晶的参数。当然, 一个人应该尝试用刚准备好的样品进行结晶, 因此相应的协议必须是直接的, 并且随时可用。
在这里, 两个全自动系统设计的 LMB (系统1和 2) 和相应的协议被充分描述。这两种系统的主要应用是在坐滴结晶板上进行蒸汽扩散初始筛选。系统1集成了一个液体处理程序, 一个自动旋转到股票板, 一个喷墨打印机的车牌标签和一个胶粘剂板封口机。在系统 1, 72 96-井板充满了商业可用的筛选套件 (80 µL 的条件转移到水库从10毫升的起始量在试管), 标签和密封。然后, 这些盘子被储存在一个10° c 的恒温箱里, 用户可以随时使用它们 (最初的屏幕叫做 "LMB 板")。
系统2集成了一个液体处理器, 一个 nanoliter 分配器, 和一个胶粘剂板封口机。在系统2上, 通过结合条件和试样在 20 48-或 96-井板预填充条件下的上部井中, 产生了蒸汽扩散实验的坐液滴 (100-1、000 nL)。这意味着在系统2上使用 20 LMB 板时, 会试用1920初始筛选条件。
机器人也被单独用于优化选定的条件, 并描述了相应的半自动化协议。通常使用4角方法17来生成优化屏幕。相应的协议首先需要手动准备4解决方案 ("A、B、C 和 D")。两个线性梯度的浓度 (两个主要结晶剂), 然后自动生成直接进入储层的结晶板。为此, 以注射器为基础的液体处理程序在不同的比率下免除了4角解。
为了进一步优化条件, 可以使用可增强结果晶体属性的加性屏幕18。有两种方法可用于加性筛选: 一种协议, 在设置液滴 (协议 1) 和附加屏幕的另一种协议开始之前, 在结晶板的储层中加入添加剂 直接放到水滴上 (协议 2)。
还提出了其他有益的发展, 在湄公河 LMB, 以促进自动化大分子结晶, 也介绍了。从根本上说, 结晶板和相关的设备, 如一个可堆叠的生物分子筛选 (SBS) 盖子, 最大限度地减少蒸发的条件时使用系统2。
为简洁起见, 假定用户熟悉 nanoliter 分配器、喷墨打印机和粘板封口机的基本功能和维护。除非另有说明, 在机器人的甲板上的板块定位, 使井 A1 ("A1-corner") 是朝向后左角的一个板块承运人。

<table><tbody><tr><td>&lt;strong&gt;Robots&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Freedom EVO&amp;reg;</td><td>Tecan</td><td>n/a</td><td>Liquid handler (System 1). Aspiration/Dispense based on system liquid. Integrates an automated carousel. EVOware plus controlling software v.2.4.12.0.&amp;nbsp;</td></tr><tr><td>Microlab&amp;reg; STAR&amp;trade;</td><td>Hamilton</td><td>n/a</td><td>Liquid handler (System 2). Aspiration/Dispense based on positive displacement (CO-RE&amp;trade; technology). Hamilton STAR controlling software v.4.3.5.4785 with method management interface.</td></tr><tr><td>Mosquito&amp;reg;</td><td>TTP Labtech</td><td>n/a</td><td>Microsyringe-based nanoliter dispenser used to set up droplets (System 2 and stand-alone), 3-position deck. Controlling software v.3.11.0.1422.&amp;nbsp;</td></tr><tr><td>Dragonfly&amp;reg;</td><td>TTP Labtech</td><td>n/a</td><td>Syringe-based liquid handler used to produce optimization screens (4-corner method). Controlling software v.1.2.1.10196.&amp;nbsp;</td></tr><tr><td>Adhesive plate sealer</td><td>Brandel</td><td>n/a</td><td>Integrated to Systems 1 and 2 (also used as stand-alone robot).</td></tr><tr><td>Inkjet printer 9232</td><td>Markem-Imaje</td><td>n/a</td><td>Integrated to System 1. Touchscreen interface.</td></tr><tr><td>&lt;strong&gt;Crystallization screens&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Crystal Screen&amp;trade; 1</td><td>Hampton Research</td><td>HR2-110</td><td>Crystallization kit (test tubes, 10mL per condition) used in LMB01</td></tr><tr><td>Crystal Screen 2&amp;trade;</td><td>Hampton Research</td><td>HR2-112</td><td>Crystallization kit (test tubes, 10mL per condition) used in LMB01</td></tr><tr><td>Wizard&amp;trade; Classic 1</td><td>Rigaku</td><td>1009530</td><td>Crystallization kit (test tubes, 10mL per condition) used in LMB02</td></tr><tr><td>Wizard&amp;trade; Classic 2</td><td>Rigaku</td><td>1009531</td><td>Crystallization kit (test tubes, 10mL per condition) used in LMB02</td></tr><tr><td>Grid Screen&amp;trade; Ammonium Sulfate</td><td>Hampton Research</td><td>HR2-211</td><td>Crystallization kit (test tubes, 10mL per condition) used in LMB03</td></tr><tr><td>Grid Screen&amp;trade; PEG/LiCl</td><td>Hampton Research</td><td>HR2-217</td><td>Crystallization kit (test tubes, 10mL per condition) used in LMB03</td></tr><tr><td>Quick Screen&amp;trade;</td><td>Hampton Research</td><td>HR2-221</td><td>Crystallization kit (test tubes, 10mL per condition) used in LMB03</td></tr><tr><td>Grid Screen&amp;trade; Sodium Chloride</td><td>Hampton Research</td><td>HR2-219</td><td>Crystallization kit (test tubes, 10mL per condition) used in LMB03</td></tr><tr><td>Grid Screen&amp;trade; PEG 6000</td><td>Hampton Research</td><td>HR2-213</td><td>Crystallization kit (test tubes, 10mL per condition) used in LMB04</td></tr><tr><td>Grid Screen&amp;trade; MPD</td><td>Hampton Research</td><td>HR2-215</td><td>Crystallization kit (test tubes, 10mL per condition) used in LMB04</td></tr><tr><td>MemFac&amp;trade;</td><td>Hampton Research</td><td>HR2-114</td><td>Crystallization kit (test tubes, 10mL per condition) used in LMB04</td></tr><tr><td>PEG/Ion&amp;trade;</td><td>Hampton Research</td><td>HR2-126</td><td>Crystallization kit (test tubes, 10mL per condition) used in LMB05</td></tr><tr><td>Natrix&amp;trade;</td><td>Hampton Research</td><td>HR2-116</td><td>Crystallization kit (test tubes, 10mL per condition) used in LMB05</td></tr><tr><td>Crystal Screen Lite&amp;trade;</td><td>Hampton Research</td><td>HR2-128</td><td>Crystallization kit (test tubes, 10mL per condition) used in LMB06</td></tr><tr><td>Custom Lite screen</td><td>Molecular Dimensions Ltd</td><td>n/a</td><td>Crystallization kit (test tubes, 10mL per condition) used in LMB06</td></tr><tr><td>Wizard&amp;trade; Cryo 1</td><td>Rigaku</td><td>1009536</td><td>Crystallization kit (test tubes, 10mL per condition) used in LMB07</td></tr><tr><td>Wizard&amp;trade; Cryo 2</td><td>Rigaku</td><td>1009537</td><td>Crystallization kit (test tubes, 10mL per condition) used in LMB07</td></tr><tr><td>JBS1</td><td>JenaBioScience</td><td>CS-101L</td><td>Crystallization kit (test tubes, 10mL per condition) used in LMB08</td></tr><tr><td>JBS2</td><td>JenaBioScience</td><td>CS-102L</td><td>Crystallization kit (test tubes, 10mL per condition) used in LMB08</td></tr><tr><td>JBS3</td><td>JenaBioScience</td><td>CS-103L</td><td>Crystallization kit (test tubes, 10mL per condition) used in LMB08</td></tr><tr><td>JBS4</td><td>JenaBioScience</td><td>CS-104L</td><td>Crystallization kit (test tubes, 10mL per condition) used in LMB08</td></tr><tr><td>JBS5</td><td>JenaBioScience</td><td>CS-105L</td><td>Crystallization kit (test tubes, 10mL per condition) used in LMB09</td></tr><tr><td>JBS6</td><td>JenaBioScience</td><td>CS-106L</td><td>Crystallization kit (test tubes, 10mL per condition) used in LMB09</td></tr><tr><td>JBS7</td><td>JenaBioScience</td><td>CS-107L</td><td>Crystallization kit (test tubes, 10mL per condition) used in LMB09</td></tr><tr><td>JBS8</td><td>JenaBioScience</td><td>CS-108L</td><td>Crystallization kit (test tubes, 10mL per condition) used in LMB09</td></tr><tr><td>JBS9</td><td>JenaBioScience</td><td>CS-109L</td><td>Crystallization kit (test tubes, 10mL per condition) used in LMB10</td></tr><tr><td>JBS10</td><td>JenaBioScience</td><td>CS-110L</td><td>Crystallization kit (test tubes, 10mL per condition) used in LMB10</td></tr><tr><td>Clear Strategy&amp;trade; Screen 1 pH 4.5</td><td>Molecular Dimensions Ltd</td><td>MD1-16LMB</td><td>Crystallization kit (test tubes, 10mL per condition) used in LMB10</td></tr><tr><td>Clear Strategy&amp;trade; Screen 1 pH 5.5</td><td>Molecular Dimensions Ltd</td><td>MD1-16LMB</td><td>Crystallization kit (test tubes, 10mL per condition) used in LMB10</td></tr><tr><td>Clear Strategy&amp;trade; Screen 1 pH 6.5</td><td>Molecular Dimensions Ltd</td><td>MD1-16LMB</td><td>Crystallization kit (test tubes, 10mL per condition) used in LMB11</td></tr><tr><td>Clear Strategy&amp;trade; Screen 1 pH 7.5</td><td>Molecular Dimensions Ltd</td><td>MD1-16LMB</td><td>Crystallization kit (test tubes, 10mL per condition) used in LMB11</td></tr><tr><td>Clear Strategy&amp;trade; Screen 1 pH 8.5</td><td>Molecular Dimensions Ltd</td><td>MD1-16LMB</td><td>Crystallization kit (test tubes, 10mL per condition) used in LMB11</td></tr><tr><td>Clear Strategy&amp;trade; Screen 2 pH 4.5</td><td>Molecular Dimensions Ltd</td><td>MD1-16LMB</td><td>Crystallization kit (test tubes, 10mL per condition) used in LMB11</td></tr><tr><td>Clear Strategy&amp;trade; Screen 2 pH 5.5</td><td>Molecular Dimensions Ltd</td><td>MD1-16LMB</td><td>Crystallization kit (test tubes, 10mL per condition) used in LMB12</td></tr><tr><td>Clear Strategy&amp;trade; Screen 2 pH 6.5</td><td>Molecular Dimensions Ltd</td><td>MD1-16LMB</td><td>Crystallization kit (test tubes, 10mL per condition) used in LMB12</td></tr><tr><td>Clear Strategy&amp;trade; Screen 2 pH 7.5</td><td>Molecular Dimensions Ltd</td><td>MD1-16LMB</td><td>Crystallization kit (test tubes, 10mL per condition) used in LMB12</td></tr><tr><td>Clear Strategy&amp;trade; Screen 2 pH 8.5</td><td>Molecular Dimensions Ltd</td><td>MD1-16LMB</td><td>Crystallization kit (test tubes, 10mL per condition) used in LMB12</td></tr><tr><td>Index&amp;trade;</td><td>Hampton Research</td><td>HR2-144</td><td>Crystallization kit (test tubes, 10mL per condition) used in LMB13</td></tr><tr><td>SaltRX&amp;trade; 1</td><td>Hampton Research</td><td>HR2-107</td><td>Crystallization kit (test tubes, 10mL per condition) used in LMB14</td></tr><tr><td>SaltRX&amp;trade; 2</td><td>Hampton Research</td><td>HR2-109</td><td>Crystallization kit (test tubes, 10mL per condition) used in LMB14</td></tr><tr><td>MemStar&amp;trade;</td><td>Molecular Dimensions Ltd</td><td>MD1-21</td><td>Crystallization kit (test tubes, 10mL per condition) used in LMB15</td></tr><tr><td>MemSys&amp;trade;</td><td>Molecular Dimensions Ltd</td><td>MD1-25</td><td>Crystallization kit (test tubes, 10mL per condition) used in LMB15</td></tr><tr><td>JCSG-plus&amp;trade; Suite</td><td>Qiagen</td><td>130720</td><td>Crystallization kit (test tubes, 10mL per condition) used in LMB16</td></tr><tr><td>MORPHEUS&amp;reg; screen</td><td>Molecular Dimensions Ltd</td><td>MD1-46</td><td>Crystallization kit (test tubes, 10mL per condition) used in LMB17</td></tr><tr><td>Pi minimal screen</td><td>JenaBioScience</td><td>CS-127</td><td>Crystallization kit (test tubes, 10mL per condition) used in LMB18</td></tr><tr><td>Pi-PEG screen</td><td>JenaBioScience</td><td>CS-128</td><td>Crystallization kit (test tubes, 10mL per condition) used in LMB19</td></tr><tr><td>MORPHEUS&amp;reg; II screen</td><td>Molecular Dimensions Ltd</td><td>MD1-91</td><td>Crystallization kit (test tubes, 10mL per condition) used in LMB20</td></tr><tr><td>LMB crystallization screen&amp;trade;</td><td>Molecular Dimensions Ltd</td><td>MD1-98</td><td>Crystallization kit (test tubes, 10mL per condition) used in LMB21</td></tr><tr><td>&lt;strong&gt;Additive screens&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>HT additive screen</td><td>Hampton Research</td><td>HR2-138</td><td>Frozen in 96-well deepwell block (1 mL per well).</td></tr><tr><td>MORPHEUS&amp;reg; additive screen</td><td>Molecular Dimensions Ltd</td><td>MD1-93-500</td><td>Frozen in 96-well deepwell block (500 &amp;micro;L per well).</td></tr><tr><td>ANGSTROM additive screen&amp;trade;&amp;nbsp;</td><td>Molecular Dimensions Ltd</td><td>MD1-100</td><td>Frozen in 96-well deepwell block (1 mL per well).</td></tr><tr><td>MORPHEUS&amp;reg; additive screen</td><td>Molecular Dimensions Ltd</td><td>MD1-93</td><td>Frozen in 96-well cell culture Costar&amp;reg; plate with V-shaped wells (100 &amp;micro;L per well).</td></tr><tr><td>ANGSTROM additive screen&amp;trade;&amp;nbsp;</td><td>Molecular Dimensions Ltd</td><td>MD1-100-FX</td><td>Frozen in 96-well cell culture Costar&amp;reg; plate with V-shaped well (100 &amp;micro;L per well).</td></tr><tr><td>HIPPOCRATES additive screen</td><td>Molecular Dimensions Ltd</td><td>n/a</td><td>48 single additives (drug compounds found in MORPHEUS&amp;reg; III).</td></tr><tr><td>&lt;strong&gt;Other consumables&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>96-well MRC 2-drop plate</td><td>Swissci</td><td>MRC 96T-UVP</td><td>Sitting-drop, vapor diffusion plate. Reservoir recommended volume: 80&amp;nbsp; &amp;micro;L. Range of useful droplet volumes: 10-1000 nL. UV transmissible.</td></tr><tr><td>48-well MRC 1-drop plate&amp;nbsp; (&#039;MAXI plate&#039;)</td><td>Swissci</td><td>MMX01-UVP&amp;nbsp;</td><td>Sitting-drop, vapor diffusion plate for scale-up/optimization. Reservoir recommended volume: 200&amp;nbsp; &amp;micro;L. Range of useful droplet volumes: 0.1-10 &amp;micro;L. UV transmissible.</td></tr><tr><td>MRC hanging drop seal</td><td>Swissci</td><td>n/a</td><td>Hanging-drop, compatible with both MRC vapor diffusion plates (MRC 96T-UVP and MMX01-UVP ). UV and X-ray transmissible.</td></tr><tr><td>Adhesive sealing tape</td><td>Hampton Research</td><td>HR4-50</td><td>3-inch wide Duck&amp;reg; HD Clear&amp;trade; for sealer and manual sealing.</td></tr><tr><td>Adhesive aluminium sheet</td><td>Beckman Coulter</td><td>538619</td><td>Used to reseal additive screens.</td></tr><tr><td>Ink cartridge</td><td>Markem-Imaje</td><td>9651</td><td>System 1 (inkjet printer).</td></tr><tr><td>Solvent cartridge</td><td>Markem-Imaje</td><td>8652</td><td>System 1 (inkjet printer).</td></tr><tr><td>50 &amp;micro;L tips&amp;nbsp;</td><td>Hamilton</td><td>235947</td><td>System 2 (STAR&amp;trade; liquid handler). Box of 6 sets with 1920 x CO-RE&amp;trade; tips in disposable stacks.</td></tr><tr><td>Reagent container</td><td>Hamilton</td><td>194052</td><td>Used to dispense a condition into plate(s) during additive screening protocols.&amp;nbsp;</td></tr><tr><td>PCR plate</td><td>Thermo Scientific&amp;trade;</td><td>AB-2150</td><td>System 2 (contains protein to be transfer to the Mosquito&amp;reg;). Abgene Diamond ultra, 384 V-shaped wells.</td></tr><tr><td>microsyringes</td><td>TTP Labtech</td><td>4150-03020</td><td>Spool of 26,000 microsyringes for the Mosquito&amp;reg; nanoliter dispenser (9mm spacing).</td></tr><tr><td>strip-holder block</td><td>TTP Labtech</td><td>3019-05013</td><td>SSB device for the Mosquito&amp;reg; strips, aka &#039;4-way Reagent Holder&#039;.</td></tr><tr><td>2 &amp;micro;L 8-well strip</td><td>TTP Labtech</td><td>4150-03110</td><td>Contains protein on the deck of the Mosquito&amp;reg;. Box of 40 strips, max. vol. in well is 3.2 &amp;micro;L.</td></tr><tr><td>5 &amp;micro;L 8-well strip</td><td>TTP Labtech</td><td>4150-03100</td><td>Contains protein on the deck of the Mosquito&amp;reg;. Box of 40 strips, max. vol. in well is 7.5 &amp;micro;L.</td></tr><tr><td>5 mL syringes</td><td>TTP Labtech</td><td>4150-07100</td><td>Syringe body and piston for the Dragonfly&amp;reg; liquid handler. Pack of 100.</td></tr><tr><td>Troughs/Reservoirs</td><td>TTP Labtech</td><td>4150-07103</td><td>Contains stock solutions on the deck of the Dragonfly&amp;reg;. Pack of 50.</td></tr><tr><td>Orbital microplate shaker</td><td>CamLab Limited</td><td>n/a</td><td>Variomag&amp;reg; for mixing conditions in a single plate (0-2000 rpm).</td></tr><tr><td>Microplate mixer</td><td>TTP Labtech</td><td>3121-01015</td><td>MxOne. Mixing condition in a single plate with 96 vibrating pins.</td></tr></tbody></table>

automated protocols for macromolecular crystallization at the mrc laboratory of molecular biology

当高质量的晶体得到衍射 X 射线时, 晶体结构可以在近原子分辨率下解决。但不能预测蛋白质、dna、rna 及其配合物的结晶条件。采用多种条件是提高质量衍射晶体成品率的一种方法。两个全自动系统已经开发了在湄公河医学实验室的分子生物学 (剑桥, 英国, 湄公河 LMB), 以促进结晶筛选的1920初始条件的蒸气扩散的 nanoliter 液滴。还开发了半自动化协议, 通过改变试剂浓度、pH 值或引入可能增强所产生晶体性能的添加剂来优化条件。所有相应的协议将被详细描述和简要讨论。两者结合在一起, 在多用户设施中实现了方便高效的大分子结晶, 同时使用户能够控制实验的关键参数。

Watch this Scientific Journal Video about Automated Protocols for Macromolecular Crystallization at the MRC Laboratory of Molecular Biology at JoVE.com

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

本文介绍和讨论了用于蒸汽扩散实验的大量屏幕和 nanoliter 结晶液滴的常规制备的自动化系统和协议。

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 ...

automated-protocols-for-macromolecular-crystallization-at-mrc

Research

JoVE Journal

Biochemistry

16.3K 次观看.  Medical Research Council. 本文介绍和讨论了用于蒸汽扩散实验的大量屏幕和 nanoliter 结晶液滴的常规制备的自动化系统和协议。

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

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 ...

科研

生物化学

Genetic Engineering of Dictyostelium discoideum Cells Based on Selection and Growth on Bacteria

Dictyostelium discoideum is an intriguing model organism for the study of cell differentiation processes during development, cell signaling, and other important cellular biology questions. The technologies available to genetically manipulate Dictyostelium cells are well-developed. Transfections can be performed using different selectable markers and marker re-cycling, including homologous recombination and insertional mutagenesis. This is supported by a well-annotated genome. However, these approaches are optimized for axenic cell lines growing in liquid cultures and are difficult to apply to non-axenic wild-type cells, which feed only on bacteria. The mutations that are present in axenic strains disturb Ras signaling, causing excessive macropinocytosis required for feeding, and impair cell migration, which confounds the interpretation of signal transduction and chemotaxis experiments in those strains. Earlier attempts to genetically manipulate non-axenic cells have lacked efficiency and required complex experimental procedures. We have developed a simple transfection protocol that, for the first time, overcomes these limitations. Those series of large improvements to Dictyostelium molecular genetics allow wild-type cells to be manipulated as easily as standard laboratory strains. In addition to the advantages for studying uncorrupted signaling and motility processes, mutants that disrupt macropinocytosis-based growth can now be readily isolated. Furthermore, the entire transfection workflow is greatly accelerated, with recombinant cells that can be generated in days rather than weeks. Another advantage is that molecular genetics can further be performed with freshly isolated wild-type Dictyostelium samples from the environment. This can help to extend the scope of approaches used in these research areas.

Genetic Engineering of Dictyostelium discoideum Cells Based on Selection and Growth on Bacteria

Dictyostelium discoideum is a popular model organism to study complex cellular processes such as cell migration, endocytosis, and development. The utility of the organism is dependent on the feasibility of genetic manipulation. Here, we present methods to transfect Dictyostelium discoideum cells that overcome existing limitations of culturing cells in liquid media.

基于细菌选择和生长的盘状细胞基因工程

Dictyostelium discoideum is an intriguing model organism for the study of cell differentiation processes during development, cell signaling, and other ...

遗传学

Single-step Purification of Macromolecular Complexes Using RNA Attached to Biotin and a Photo-cleavable Linker

For many years, the exceptionally strong and rapidly formed interaction between biotin and streptavidin has been successfully utilized for partial purification of biologically important RNA/protein complexes. However, this strategy suffers from one major disadvantage that limits its broader utilization: the biotin/streptavidin interaction can be broken only under denaturing conditions that also disrupt the integrity of the eluted complexes, hence precluding their subsequent functional analysis and/or further purification by other methods. In addition, the eluted samples are frequently contaminated with the background proteins that nonspecifically associate with streptavidin beads, complicating the analysis of the purified complexes by silver staining and mass spectrometry. To overcome these limitations, we developed a variant of the biotin/streptavidin strategy in which biotin is attached to an RNA substrate via a photo-cleavable linker and the complexes immobilized on streptavidin beads are selectively eluted to solution in a native form by long wave UV, leaving the background proteins on the beads. Shorter RNA binding substrates can be synthesized chemically with biotin and the photo-cleavable linker covalently attached to the 5&#39; end of the RNA, whereas longer RNA substrates can be provided with the two groups by a complementary oligonucleotide. These two variants of the UV-elution method were tested for purification of the U7 snRNP-dependent processing complexes that cleave histone pre-mRNAs at the 3&#39; end and they both proved to compare favorably to other previously developed purification methods. The UV-eluted samples contained readily detectable amounts of the U7 snRNP that was free of major protein contaminants and suitable for direct analysis by mass spectrometry and functional assays. The described method can be readily adapted for purification of other RNA binding complexes and used in conjunction with single- and double-stranded DNA binding sites to purify DNA-specific proteins and macromolecular complexes.

RNA/protein complexes purified using botin-streptavidin strategy are eluted to solution under denaturing conditions in a form unsuitable for further purification and functional analysis. Here, we describe a modification of this strategy that utilizes a photo-cleavable linker in RNA and a gentle UV-elution step, yielding native and fully functional RNA/protein complexes.

生物素和光裂解链接器所附 rna 对高分子配合物的单步纯化

For many years, the exceptionally strong and rapidly formed interaction between biotin and streptavidin has been successfully utilized for partial ...

A Behavioral Assay for Investigating the Role of Spatial Memory During Instinctive Defense in Mice

Evolution has selected a repertoire of defensive behaviors that are essential for survival across all animal species. These behaviors are often stereotyped actions elicited in response to innately aversive sensory stimuli, but their success requires enough flexibility for adapting to different spatial environments, which can change rapidly. Here, we describe a behavioral assay to evaluate the influence of learned spatial knowledge on defensive behaviors in mice. We have adapted the widely used Barnes maze spatial memory assay to investigate how mice navigate to a shelter during escape responses to innately aversive sensory stimuli in a novel environment, and how they adapt to acute changes in the environment. This new assay is an ethological paradigm that does not require training and exploits the natural exploration patterns and navigation strategies in mice. We propose that the set of protocols described here are a powerful means of studying goal-directed behaviors and stimulus-triggered navigation, which should be of interest to both the fields of instinctive behaviors and spatial memory.

This protocol describes a behavioral assay, based on the Barnes maze test, to study how instinctive defensive actions are modified by the knowledge of spatial environment.

一种研究空间记忆在小鼠本能防御中作用的行为分析法

Evolution has selected a repertoire of defensive behaviors that are essential for survival across all animal species. These behaviors are often ...

行为学

Proteome-wide Quantification of Labeling Homogeneity at the Single Molecule Level

Cell proteomes are often characterized using electrophoresis assays, where all species of proteins in the cells are non-specifically labeled with a fluorescent dye and are spotted by a photodetector following their separation. Single molecule fluorescence imaging can provide ultrasensitive protein detection with its ability for visualizing individual fluorescent molecules. However, the application of this powerful imaging method to electrophoresis assays is hampered by the lack of ways to characterize the homogeneity of fluorescent labeling of each protein species across the proteome. Here, we developed a method to evaluate the labeling homogeneity across the proteome based on a single molecule fluorescence imaging assay. In our measurement using a HeLa cell sample, the proportion of proteins labeled with at least one dye, which we termed &#8216;labeling occupancy&#8217; (LO), was determined to range from 50% to 90%, supporting the high potential of the application of single molecule imaging to sensitive and precise proteome analysis.

Here, we present a protocol to assess the labeling homogeneity for each protein species in a complex protein sample at the single molecule level.

单分子水平标记均匀性的全蛋白质定量

Cell proteomes are often characterized using electrophoresis assays, where all species of proteins in the cells are non-specifically labeled with a ...

Characterization at the Molecular Level using Robust Biochemical Approaches of a New Kinase Protein

Extensive whole genome sequencing has identified many Open Reading Frames (ORFs) providing many potential proteins. These proteins may have important roles for the cell and may unravel new cellular processes. Among proteins, kinases are major actors as they belong to cell signaling pathways and have the ability to switch on or off many processes crucial to the fate of the cell, such as cell growth, division, differentiation, motility, and death.
In this study, we focused on a new potential kinase protein, LIMK2-1. We demonstrated its existence by Western Blot using a specific antibody. We evaluated its interaction with an upstream regulating protein using coimmunoprecipitation experiments. Coimmunoprecipitation is a very powerful technique able to detect the interaction between two target proteins. It may also be used to detect new partners of a bait protein. The bait protein may be purified either via a tag engineered to its sequence or via an antibody specifically targeting it. These protein complexes may then be separated by SDS-PAGE (Sodium Dodecyl Sulfate PolyAcrylamide Gel) and identified using mass spectrometry. Immunoprecipitated LIMK2-1 was also used to test its kinase activity in vitro by &#947;[32P] ATP labeling. This well-established assay may use many different substrates, and mutated versions of the bait may be used to assess the role of specific residues. The effects of pharmacological agents may also be evaluated since this technique is both highly sensitive and quantitative. Nonetheless, radioactivity handling requires particular caution. Kinase activity may also be assessed with specific antibodies targeting the phospho group of the modified amino acid. These kinds of antibodies are not commercially available for all the phospho modified residues.

We characterized a new kinase protein using robust biochemical approaches: Western Blot analysis with a dedicated specific antibody on different cell lines and tissues, interactions by coimmunoprecipitation experiments, kinase activity detected by Western Blot using a phospho-specific antibody and by &#947;[32P] ATP labeling.

使用新激酶蛋白的强生化方法在分子水平上进行表征

Extensive whole genome sequencing has identified many Open Reading Frames (ORFs) providing many potential proteins. These proteins may have important ...

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

The key to determining crystal structures of membrane protein complexes is the quality of the sample prior to crystallization. In particular, the choice of detergent is critical, because it affects both the stability and monodispersity of the complex. We recently determined the crystal structure of an active state of bovine rhodopsin coupled to an engineered G protein, mini-Go, at 3.1 &#197; resolution. Here, we detail the procedure for optimizing the preparation of the rhodopsin&#8211;mini-Go complex. Dark-state rhodopsin was prepared in classical and neopentyl glycol (NPG) detergents, followed by complex formation with mini-Go under light exposure. The stability of the rhodopsin was assessed by ultraviolet-visible (UV-VIS) spectroscopy, which monitors the reconstitution into rhodopsin of the light-sensitive ligand, 9-cis retinal. Automated size-exclusion chromatography (SEC) was used to characterize the monodispersity of rhodopsin and the rhodopsin&#8211;mini-Go complex. SDS-polyacrylamide electrophoresis (SDS-PAGE) confirmed the formation of the complex by identifying a 1:1 molar ratio between rhodopsin and mini-Go after staining the gel with Coomassie blue. After cross-validating all this analytical data, we eliminated unsuitable detergents and continued with the best candidate detergent for large-scale preparation and crystallization. An additional problem arose from the heterogeneity of N-glycosylation. Heterologously-expressed rhodopsin was observed on SDS-PAGE to have two different N-glycosylated populations, which would probably have hindered crystallogenesis. Therefore, different deglycosylation enzymes were tested, and endoglycosidase F1 (EndoF1) produced rhodopsin with a single species of N-glycosylation. With this strategic pipeline for characterizing protein quality, preparation of the rhodopsin&#8211;mini-Go complex was optimized to deliver the crystal structure. This was only the third crystal structure of a GPCR&#8211;G protein signaling complex. This approach can also be generalized for other membrane proteins and their complexes to facilitate sample preparation and structure determination.

This report describes screening of different detergents for preparing the visual GPCR, rhodopsin, and its complex with mini-Go. Biochemical methods characterizing the quality of the complex at different stages during purification are demonstrated. This protocol can be generalized to other membrane protein complexes for their future structural studies.

视觉GPCR-迷你G蛋白信号复合物的战略筛选与表征，成功结晶

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

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and the inherent instability of many membrane proteins once solubilized in detergents. A protocol is described that combines ligation independent cloning of membrane proteins as GFP fusions with expression in Escherichia coli detected by GFP fluorescence. This enables the construction and expression screening of multiple membrane protein/variants to identify candidates suitable for further investment of time and effort. The GFP reporter is used in a primary screen of expression by visualizing GFP fluorescence following SDS polyacrylamide gel electrophoresis (SDS-PAGE). Membrane proteins that show both a high expression level with minimum degradation as indicated by the absence of free GFP, are selected for a secondary screen. These constructs are scaled and a total membrane fraction prepared and solubilized in four different detergents. Following ultracentrifugation to remove detergent-insoluble material, lysates are analyzed by fluorescence detection size exclusion chromatography (FSEC). Monitoring the size exclusion profile by GFP fluorescence provides information about the mono-dispersity and integrity of the membrane proteins in different detergents. Protein: detergent combinations that elute with a symmetrical peak with little or no free GFP and minimum aggregation are candidates for subsequent purification. Using the above methodology, the heterologous expression in E. coli of SED (shape, elongation, division, and sporulation) proteins from 47 different species of bacteria was analyzed. These proteins typically have ten transmembrane domains and are essential for cell division. The results show that the production of the SEDs orthologues in E. coli was highly variable with respect to the expression levels and integrity of the GFP fusion proteins. The experiment identified a subset for further investigation.

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

A streamlined approach to screening for the expression of recombinant membrane proteins in Escherichia coli based on fusion to green fluorescent protein is presented.

膜蛋白的绿色荧光蛋白为基础的表达筛选<em&gt;大肠杆菌</em

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and ...

生物学

Flexible Measurement of Bioluminescent Reporters Using an Automated Longitudinal Luciferase Imaging Gas- and Temperature-optimized Recorder (ALLIGATOR)

Luciferase-based reporters of cellular gene expression are in widespread use for both longitudinal and end-point assays of biological activity. In circadian rhythms research, for example, clock gene fusions with firefly luciferase give rise to robust rhythms in cellular bioluminescence that persist over many days. Technical limitations associated with photomultiplier tubes (PMT) or conventional microscopy-based methods for bioluminescence quantification have typically demanded that cells and tissues be maintained under quite non-physiological conditions during recording, with a trade-off between sensitivity and throughput. Here, we report a refinement of prior methods that allows long-term bioluminescence imaging with high sensitivity and throughput which supports a broad range of culture conditions, including variable gas and humidity control, and that accepts many different tissue culture plates and dishes. This automated longitudinal luciferase imaging gas- and temperature-optimized recorder (ALLIGATOR) also allows the observation of spatial variations in luciferase expression across a cell monolayer or tissue, which cannot readily be observed by traditional methods. We highlight how the ALLIGATOR provides vastly increased flexibility for the detection of luciferase activity when compared with existing methods.

Flexible Measurement of Bioluminescent Reporters Using an Automated Longitudinal Luciferase Imaging Gas- and Temperature-optimized Recorder ALLIGATOR

Genetically encoded luciferase is a popular non-invasive reporter of gene expression. Use of an automated longitudinal luciferase imaging gas- and temperature-optimized recorder (ALLIGATOR) enables longitudinal recording from bioluminescent cells under a wide range of conditions. Here we show how ALLIGATOR may be used in the context of circadian rhythms research.

利用自动纵向荧光成像气体和温度优化记录仪 (鳄鱼) 对生物发光记者进行灵活测量

Luciferase-based reporters of cellular gene expression are in widespread use for both longitudinal and end-point assays of biological activity. In ...

研究 ATG9A 在细胞中的定位

Imaging ATG9A, a Multi-Spanning Membrane Protein

Autophagy is a highly conserved pathway that the cell uses to maintain homeostasis, degrade damaged organelles, combat invading pathogens, and survive pathological conditions. A set of proteins, called ATG proteins, comprise the core autophagy machinery and work together in a defined hierarchy. Studies in recent years have improved our knowledge of the autophagy pathway. Most recently, it has been proposed that ATG9A vesicles are at the heart of autophagy, as they control the rapid de novo synthesis of an organelle called the phagophore. The study of ATG9A has proven challenging, since ATG9A is a transmembrane protein, and it is present in different membrane compartments. As such, understanding its trafficking is an important element for understanding autophagy. Here, detailed methods are presented that can be used to study ATG9A and, in particular, its localization using immunofluorescence techniques, which can be assessed and quantified. The pitfalls of transient overexpression are also addressed. The correct characterization of ATG9A function and the standardization of techniques to analyze its trafficking are crucial to further characterize the events governing autophagy initiation.

作者聚焦:成像 ATG9A，一种多跨膜蛋白  

This protocol describes various methods that can help in the study of ATG9A biology, including immunofluorescence followed by image analysis, transient overexpression considerations, and investigating the ATG9A glycosylation status using western blot.

成像 ATG9A，一种多跨膜蛋白

Autophagy is a highly conserved pathway that the cell uses to maintain homeostasis, degrade damaged organelles, combat invading pathogens, and survive ...