我们感谢美国地质调查哈蒙德湾生物站的使用他们的研究设施和工作人员的美国鱼类和野生动物服务和渔业和海洋加拿大提供海洋鳗。这项研究得到了大湖渔业委员会给予未明李和科李的赠款的支持。

这里描述的所有方法都已获得密歇根州立大学机构动物保育和使用委员会 (AUF 03/14-054-00 和 02/17-031-00) 的批准。
1. 海洋鳗条件水的收集和提取
<ol><li>安置性成熟男性海鳗 (15-30 个动物) 在供应与 250 L 的被加气的湖休伦的水被维护在 16-18 °c 的坦克。</li>
	<li>每晚从琼到7月收集男性条件的水。 注: 海洋鳗产卵后自然死亡。如果一条鱼在它的生命中接近这一点, 用新鲜成熟的雄性代替它。</li>
	<li>固相萃取萃取条件水。<ol>
<li>将树脂浸泡在甲醇中4小时, 然后将其装入柱内。将树脂装入柱内, 然后泵入10升水通过柱, 消除有机溶剂。</li>
		<li>将有条件的水从水箱中通过泵浦系统传递到立柱顶部。水通过2公斤的中等极性聚合物离子交换树脂 (阳离子交换树脂 XAD-7 HP 树脂) 的床, 包含在一系列四 2.5 L 容量玻璃专栏。保持负载速度介于400和 600 mL/6 洗脱的代谢产物中, 甲醇的10升, 紧接着是丙酮的5升。 注意: 此步骤使用甲醇和丙酮。两者都是易燃和有毒的。 注: 池内残留物可存储在-80 °c, 直到进一步处理。</li>
	</ol>
</li>
	<li>在1周的浓缩后重新激活柱子, 用清水冲洗 (大约10升)。</li>
	<li>除去有机溶剂 (甲醇和水的混合物), 并在减压 (300 毫巴) 40 摄氏度的情况下, 以旋转蒸发的方法收获萃取液5小时。用冷冻干燥机将48小时的冻干干燥浓缩到干燥的-20 摄氏度。</li>
</ol>2. 用色谱法分离分数池
<ol><li>在初始移动阶段浸泡硅胶 (230-400 目), 30 分钟. 将凝胶与溶剂 (悬浮液) 转移到玻璃柱上。打开该列的阀门, 以允许移动阶段通过。允许硅胶沉淀, 形成硅胶床。</li>
	<li>将萃取物与硅胶 (70-230 目) 彻底混合, 将其装入硅胶床上的液相色谱柱上。洗脱他们与一个梯度从 95% CHCl3 (氯仿)/甲醇 (甲醇) 到100% 甲醇, 2.5 L 在总容量。收集淋洗在单独的瓶子 (每10毫升)。 注意: 这个步骤中使用的氯仿是一种有毒的试剂。</li>
	<li>用薄层色谱 (TLC) 分析方法将 eluents 的聚集性引导成20个组分。<ol>
<li>对预涂硅胶板进行薄层色谱实验, 将试样应用于起始线上, 将板材的起始线浸入显影溶剂中 (在密封玻璃中选择极性以 CHCl3与甲醇的比值为 100-0%)坦克。</li>
		<li>选择发展溶剂的比例, 使所有的薄层斑点与保留因子 (Rf) 范围从 0.3-0.8 不等。</li>
		<li>开发后的溶剂到达终点线后, 取出罐中的盘子, 等待10分钟的溶剂蒸发。</li>
		<li>在紫外线照射下, 先将斑点想象成 254 nm, 然后用一个色谱喷雾剂喷洒5% 茴香醛 (20 ul) 酸性甲醇溶液, 然后将它们加热到85摄氏度, 3 分钟。 注意: 薄层色板上的彩色斑点表示分数中的主要成分。</li>
		<li>根据专色和主成分的 Rf相似性, 将淋洗与20分数结合起来。</li>
	</ol>
</li>
	<li>在减少压力下 (300 毫巴根据溶剂的性质) 将分数集中到残留物上, 在40摄氏度左右30分钟。</li>
</ol>3. 电 olfactogram (EOG) 记录, 以识别气味的分数/化合物
<ol><li>拉硼玻璃毛细血管 (外径: 1.5 毫米; 内径: 0.86 毫米; 长度: 100 毫米), 与加热器水平设置为65的微拉出器。</li>
	<li>评分和削减开口在毛细管与钻石尖玻璃切割机, 并填补它与熔融0.4% 琼脂0.9% 盐水。毛细管尖端的开口应该直径约为10µm。</li>
	<li>填充从步骤 3.1-3.2 和固态电极持有人预先捏造与银/AgCl 丸 (见材料表) 与3米氯化钾使用微的拉毛毛细管电极。 注: 去除毛细管或电极支架内的气泡。</li>
	<li>将被拉电极插入电极夹中。</li>
	<li>在100毫升 10-5米 l-精氨酸在木炭过滤水从 10-2米精氨酸库存溶液在去离子水 (储存在4°c) 在一个容积烧瓶。将 10-5米精氨酸的20毫升转移到玻璃瓶中。</li>
	<li>对于浓度-反应曲线, 准备10毫升10倍稀释的分数池从步骤2.3 的玻璃瓶。在实验前每天准备新鲜稀释, 并在一天内使用。将 l-精氨酸和分数池稀释的工作溶液的玻璃瓶放在循环水浴中, 使温度平衡到8摄氏度。</li>
	<li>麻醉鳗与 3-苯甲酸酸乙酯 (MS222; 100 毫克/升), 并固定它与肌肉注射 gallamine triethiodide (3 毫克/公斤体重, 0.9% 生理盐水) 与无菌注射器。 注: 充分的麻醉深度是通过观察没有鳃运动, 不能保持直立姿势, 并没有吸入到坦克两侧使用其口服盘来确定的。在手术前和术中尽量减少任何微生物污染, 在使用前至少10分钟, 在70% 乙醇 (v: v) 中用无菌手套浸泡解剖工具。</li>
	<li>将麻醉鳗定向到 V 形支架上, 用湿纸巾包裹, 防止脱水。 注意: 不要阻碍鳃开口。</li>
	<li>将 MS222 50 毫克/升的循环加气水管插入颊腔内, 调整流速, 确保水通过鳃孔流出, 持续灌溉鳃。</li>
	<li>使用无菌手术刀和钳 [在立体显微镜下的1.25X 放大 (见材料表)], 以消除嗅囊表面的皮肤5毫米2切片, 以暴露嗅觉上皮。</li>
	<li>用过滤过的水冲洗气味输送管, 并将其连接到安装在机器人上的阀门上。将气味输送毛细管插入嗅上皮腔内, 使用机器人将过滤后的水输送到嗅觉上皮, 以防止不气味时干燥。</li>
	<li>在并联上安装录音和参考电极。将参考电极降低到鼻孔附近的外部皮肤。使用立体显微镜 (1.25X), 降低记录电极, 勉强触及嗅上皮的表面。</li>
	<li>将气味输送管的吸收从背景滤水转移到 10-5米精氨酸溶液中。</li>
	<li>打开计算机、放大器 (设置为 DC 模式)、滤镜和数字化器。使用阀门驱动程序软件, 通过检查 T1 盒, 设置 T 为 4, 并检查 T2 盒, 程序来管理 4 s 单脉冲气味。</li>
	<li>在数据采集软件 (见材料表) 中, 将采集模式设置为高速示波器, 单击播放图标, 然后单击启动阀驱动程序触发气味脉冲。 注: 数据采集软件自动记录二十年代的差分 EOG 响应振幅 (3 秒之前, 4 秒在气味脉冲和十三年代之后)。使用机器人来操纵记录电极、参考电极或气味输送管的位置, 以提高信噪比, 最大程度地响应 l-精氨酸标准, 对空白控制的响应最小 (过滤水)。在移动电极时, 通过将交流-直流放大器开关转换为接地, 使放大器接地。</li>
	<li>开始记录空白控制, l-精氨酸, 然后气味准备在步骤3.6 从低到高浓度和2分钟冲洗的过滤水在应用程序之间。</li>
	<li>在记录对所有要测试的气味的响应后, 接地放大器, 并仔细地收回电极和气味送毛管。继批准的机构动物护理和使用委员会 (IACUC) 方法后, 在 EOG 实验的终止, 弄死麻醉鳗的过量 MS222 (1 克/升)。 注: 检查成功的安乐死, 注意到没有鳃运动和心跳至少5分钟, 其次是 pithing 大脑。</li>
	<li>使用分析软件分析和绘制数据。确定检测气味22的阈值, 以确定引起反应大于空白水控制的气味分数。在行为分析 (步骤 4) 中, 可以得出不同于空白水控制的与浓度相关的嗅觉反应的分数池。</li>
</ol>4. 两种选择迷宫行为生物鉴定行为上活性组分/化合物
<ol><li>在迷宫的释放笼中适应性成熟的女性海鳗 (见补充图 1), 以5分钟的速度维持迷宫中河水的流速 注: 迷宫是由涂漆的海洋级木材建造的, 测量长度为6.5 米, 宽度为1.2 米, 分压器长度为2.7 米, 在上游端将迷宫分隔成两个通道。水暂时改道进入迷宫。水深应保持在0.19 米, 速度应保持在0.07 米/秒0.01。</li>
	<li>释放海洋鳗, 记录鳗在实验和控制通道中每含河水10分钟的累计时间。 注: 如果海鳗在10分钟内未能进入实验和控制通道, 则结束试验, 因为这是不活动或强侧偏倚的征兆。</li>
	<li>应用试验刺激 (即10-12 M 溶解在50% 甲醇/去离子水中的一种假定的信息素) 到随机分配的实验通道和车辆 (50% 甲醇/去离子水) 到控制通道使用蠕动泵的恒定速率为200毫升/分5分钟。 注: 测试刺激的检测阈值浓度与电 olfactogram 记录的确定应作为初始浓度的行为测试。</li>
	<li>将测试刺激和车辆加10分钟, 记录鳗在实验和控制通道中所花费的累计时间。</li>
	<li>下一次试验开始前, 用清水冲洗迷宫10分钟。如果有足够的测试刺激, 重复步骤 4.1-4.4, 至少有7鳗。</li>
	<li>计算每个试验的偏好22的索引, 并使用魏氏签名秩测试来评估其意义。 注意: 索引会产生一个数字, 可以是正数或负数。偏好指数的正值表示吸引力, 而偏好指数的负值表明排斥。如果首选项的索引与零显著不同, 则该分数被认为是活动的。 <img alt="Equation" src="/files/ftp_upload/58059/58059eq1v2.jpg"> 这里 Bc = 测试鳗在气味应用程序之前的控制通道中所花费的时间, 气味应用之前在实验频道所花费的时间, c = 在气味应用程序后的控制通道中所花费的时间, 以及 e = 气味应用程序后在实验通道中花费的时间。</li>
</ol>5. 活性馏分中纯化合物的色谱分离
<ol><li>重复步骤 2.1-2.4 与诱导嗅觉反应的分数池 (步骤 3) 和引出行为反应 (步骤 4)。</li>
	<li>用葡 LH-20 柱进一步将活性馏分提纯成具有大小排斥色谱的化合物。<ol>
<li>在初始移动相 [CHCl3-甲醇 (1:1) 或甲醇 100%] 中准备0.5 毫升的样品, 加载到相应的列 a CHCl3-甲醇 (1:1) 列, 然后是甲醇 (100%) 列, 并洗脱它们产生化合物。</li>
	</ol>
</li>
</ol>6. 纯质谱 (MS) 和核磁共振 (NMR) 的结构澄清
<ol><li>在最初的流动阶段 (通常, 甲醇到水, 1:1, v: v) 溶解和稀释高效液相色谱 (HPLC) 的纯化化合物, 形成 10 ul 溶液约1微克/毫升。<ol>
<li>将样品溶液转移到高效液相色谱瓶中, 并将其 autosampler。将样品 (10 ul) 注入电喷雾电离质谱仪 (ESIMS) 中, 用色谱质量光谱仪23记录高分辨电喷雾质谱 (HRESIMS) 谱。</li>
	</ol>
</li>
	<li>在质谱仪软件中根据数据库预测分子公式 (见材料表)24。<ol>
<li>通过选择色谱峰, 打开注射样品的色谱, 获得其质谱。</li>
		<li>将所测量的质量输入到刀具模块下元素组成中的离子 (m-/z) 值 (4 个小数)。将公差参数设置为 PPM < 5。</li>
		<li>调整符号参数, 以适应被测分子的元素组成。该软件对基于单质量分析的分子公式进行了预测。</li>
	</ol>
</li>
	<li>根据协议25 , 选择氘溶剂 (600 ul, CH3OHd4或亚砜-d6) 来溶解样品。<ol>
<li>在选定的氘溶剂中完全溶解样品, 形成溶液, 浓度约从0.1 到10毫克/毫升不等。将样品溶液转换成核磁共振管以记录 1D (1小时、 13C) 和2D 核磁共振 [1h-1H 相关光谱学 (舒适性)、异单量子相干性 (HSQC)、异多键相关性(HMBC)]光谱在900兆赫核磁共振光谱仪26。</li>
	</ol>
</li>
	<li>将核磁共振管与样品放在微调涡轮内。使用深度测量仪确保样品高度在测量窗口的中间。打开核磁共振软件 (见材料表), 点击升降机按钮, 改变磁铁中的样品。 注: 握住磁铁顶部的一只手, 感觉到来自磁铁顶部的气体。同时, 吹口哨的声音应该是发声的。</li>
	<li>轻轻地将样品放在磁铁顶部的气垫上, 再按下升降机按钮, 将样品降落到核磁共振磁体中。 注: 侦听 "单击" 噪音以指示示例处于正确的位置。</li>
	<li>创建新数据集并加载核磁共振仪器推荐的默认参数 (见材料表)。<ol>
<li>键入 "锁定" 以调用自动锁定过程, 并在提示页上选择溶剂。</li>
		<li>要进行微调, 请在 "操作" 面板中, 调整解锁模型中的按钮, 调整操作面板上的光标, 然后再次锁定磁铁。</li>
		<li>在 "提示Atma " 页上, 将 "操作" 面板中的参数调整为优化过程, 这可能需要几分钟时间。请稍候, 直到调整完成才能继续。</li>
		<li>在提示中键入 "topshim", 启动自动匀例程。等到匀完成后再继续。</li>
		<li>键入 "rga" 设置自动接收增益调整, 然后键入 "d1" 设置脉冲之间的延迟, 然后键入 "zg" 开始采集, 并等到采集完成。</li>
		<li>键入 "ef" 或 "efp" 来处理数据, 然后键入 "apk" 自动分阶段。处理核磁共振处理软件上的数据。</li>
	</ol>
</li>
	<li>通过对核磁共振数据分析的解释, 阐明化学结构。</li>
	<li>计算13C 核磁共振谱中的碳信号。从高分辨率质谱 (HR) 的预测结果中选择匹配的分子公式。</li>
	<li>将质子信号集成在1h-核磁共振谱中, 并根据 HSQC 光谱中的信号分配碳和质子的连通性。</li>
	<li>根据1小时-1小时舒适和 HMBC 谱的相关关系, 分配碳骨架的连通性。</li>
	<li>根据结构原理, 初步分配化学结构27。在化学结构数据库中搜索初步结构。将暂定结构与参照中的类似物进行比较。</li>
	<li>用核 Overhauser 效应光谱学 (NOESY) 谱分配相对构型。 注: 由于在光谱和光谱方法上显示的对映体的身份属性相同, 某些化合物的绝对构型, 特别是 2 '-酒精和 2 '-NH2, 必须用衍生反应。在衍生反应之后, 在光谱上表示的差异促进了大多数化合物的绝对构型的明确赋值28。</li>
</ol>7. EOG 和生物鉴定确认纯化合物有异味和行为上活性
<ol><li>重复步骤 3.1-3.5。</li>
	<li>对于浓度-反应曲线, 准备10毫升10倍稀释的纯化合物从 10-6米-10-13 m 的 10-3米信息素库存解决方案, 50% 甲醇/水存储在-20 °c 基于分子量确定的步骤6.2。将玻璃瓶与 l-精氨酸和纯化合物的工作溶液放在循环水浴中, 使温度平衡到8摄氏度。</li>
	<li>重复步骤 3.7-3.18。</li>
	<li>重复步骤 4.1-4.2。</li>
	<li>将纯化合物在 EOG 检测阈值集中到随机分配的实验通道和车辆 (50% 甲醇/去离子水) 到控制通道, 使用蠕动泵, 恒定率为200毫升/分钟5分钟。</li>
	<li>重复步骤 4.4-4.6。</li>
</ol>

<ol>
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作者没有什么可透露的。

鱼类生活在一个充满化合物的化学世界中, 但尚未确定。生物鉴定-引导分馏已证明是必不可少的识别和表征活性分子, 调解许多化学相互作用, 如观察鲑鲑鱼31, 亚洲大象32, 海鳗33, 34,35。生物测定引导分馏是一种有效的方法, 可以准确地从起始提取物到纯化活性化合物中精确地跟踪和定位活性物质。使用这种方法, 鉴定出来的生物活性化合物可以揭示一种新的化合物, 具有独特的化学骨架, 这是不可能从已知的合成途径预测。
EOG 记录, 以确定哪些分数或化合物诱发嗅觉反应。有几个技术上的考虑是必要的, 以准确测量的嗅觉反应对假定的费洛蒙与 EOG 录音。首先, 按照机构动物照料和使用委员会批准的程序, 鱼必须被深深麻醉与 3-苯甲酸酸乙酯 (MS222) 和固定化的肌肉注射 gallamine triethiodide。记录电极将检测到鳃和心跳的任何运动, 因为固定性不足, 这可能是电噪声的来源。第二, 在解剖后, 嗅上皮应立即暴露在木炭过滤的水中, 以防止脱水。调整记录和参考电极与并联、电场和气味输送管的位置, 可以帮助最大程度地提高对正控制气味的响应, 同时尽量减少对空白控制的响应 (过滤水)。第三, 在辨别嗅觉上皮中敏感的记录位置后, 从薄片上的类似位置记录, 以尽量减少变异是很重要的。要始终如一地记录从相似的位置, 保持坦克和 V 形塑料支架持有鱼, 显微镜, 气味输送管, 和并联在同一位置。在实验期间, 麻醉管必须留在鱼的颊腔内, 以确保其保持麻醉。然而, 如果记录电极检测到水的运动导致电信号的不稳定基线, 则在颊腔内放置和麻醉的流速可以被修改。
行为分析的设计应适应测试主体的行为生态学和兴趣研究问题。两个选择迷宫行为分析是用来确定是否有任何气味的分数也行为上活跃。由于纯化的化合物通常只能以微小的数量提供, 因此迷宫是一种有益的行为生物测定, 比流由于较低的放电。我们感兴趣的是评估的近源性信息素的偏好, 由成熟的男性发布的预测, 以吸引成熟的女性伴侣接近接近, 并保留他们在巢产卵。行为分析的目的是复制在成熟雄性气味之间的排卵雌性选择的自然条件。因此, 在我们的行为实验中, 排卵女性作为测试对象是相关的。然而, 根据气味测试, 其他测试对象 (即不同的生命阶段或男性) 可能更合适。根据测试对象的大小和信息素的预测活动空间 (即近源与远距离)36, 可能需要对两个选择迷宫的尺寸进行变化。同样, 行为反应往往是集中依赖性。如果在 EOG 确定的检测阈值上应用假定的信息素时不观察行为反应, 则应调整信息素的浓度。然而, 应该指出的是, 即使化合物是一个强有力的气味, 它可能并不一定会引起一个重要的行为偏好。最终, 在迷宫中观察到的行为反应应在与合成化合物的野外环境中进行验证, 以确认所假定的信息素的功能。
生物测定引导分馏的一个主要限制是对生物测定 (EOG 或行为) 中单个分数或化合物的顺序测试。以前的工作已经表明, 昆虫性信息素通常是多组分的混合物, 在特定比率37独立作为组分38或协同作用作为混合物39诱导适当的反应在conspecifics。因此, 只有在特定的混合物中存在的化合物, 可以忽略与生物测定引导分馏, 因为它们需要组合测试, 以确认活性。生物测定导向分馏的其他局限性包括: 1) 需要大量的起始材料, 以获得足够的结构分析、EOG 和行为测定的数量;2) 该过程由于迭代纯化过程而耗费时间;3) 不太可能检测到微小或不稳定的化合物。今后, 克服生物测定引导分馏的一些技术局限性, 可能需要采用生物测定-引导分馏和新陈代谢的杂交方法。使用杂交方法, 添加剂或协同信息素效应更可能被发现13 , 不稳定的化合物更有可能被检测到。
描述的生物测定引导分馏过程是专门为确定海洋鳗信息素而设计的。然而, 在动物王国1 , 化学通讯无处不在, 这一过程可以很容易地适应在广泛的分类群中描述信息素。信息素的识别和表征是很重要的, 因为费洛蒙可以用来调节行为反应, 从而控制入侵物种或恢复濒危本土物种。

信息素是个人发布的特定化学信号, 帮助他们找到食物来源, 检测捕食者, 并调解 conspecifics1的社会互动。昆虫信息素的传播研究已有2;然而, 水生脊椎动物信息素的化学鉴定和生物学功能还没有得到广泛的研究。对所释放的信息素的特性和功能的了解可以应用于促进被威胁物种3、4或控制害虫物种5、6的恢复。这些技术的应用需要对生物活性素成分进行分离和表征。
信息素识别是天然产物化学的一个分支。由于信息素分子本身的性质, 信息素研究的进展受到部分限制。信息素往往不稳定, 很少释放, 只有少数抽样技术存在, 以检测微量的挥发性7,8或水溶性化合物9。识别信息素的方法包括 1) 目标筛选已知化合物, 2) 新陈代谢和 3) 生物测定引导分馏。对已知化合物进行有针对性的筛选, 可以检验被推测为费洛蒙功能的生理过程的商业性代谢副产品。这种方法是有限的, 因为研究人员只能测试已知和可用的化合物。然而, 它导致了在金鱼中的性激素的成功识别功能作为费洛蒙10,11,12。新陈代谢是第二种信息素识别方法, 它区分了生物系统中潜在的小分子代谢产物13。对两组代谢剖面 (即活性与非活性提取物) 进行比较, 可以识别出代谢产物纯化的潜在新陈代谢剖面, 阐明结构, 以及生物活性证实14。复杂配方的添加剂或协同效应更可能被发现与新陈代谢, 因为代谢物被考虑在一起, 而不是作为一系列的分数13。然而, 新陈代谢的实施依赖于合成参照的可用性, 因为由此产生的数据不利于澄清新结构。
生物鉴定-引导分馏是一个综合的, 迭代的方法, 跨越两个领域: 化学和生物学。该方法利用生理和行为生物鉴定的结果来指导活性信息素化合物的分离和鉴定。粗萃取物由化学性质 (即分子量、极性等) 分馏, 并用电 olfactogram (EOG) 记录和/或在生物测定中进行测试。通过重复这些步骤的分馏和 EOGs 和/或生物鉴定, 筛选出活性成分。用光谱和光谱方法阐明了纯活性化合物的结构, 提供了分子量和结构信息, 生成了合成的模板。生物测定引导分馏可以产生不同的代谢物和潜在的新的信息素与独特的化学骨骼, 是不可能预测的生物合成途径。
在这里, 我们描述了用于分离和表征雄性海鳗性信息素化合物活性的生物测定制导分馏协议。海鳗 (Petromyzon marinus) 是研究信息素传播的理想脊椎动物模型, 因为这些鱼类大量依赖化学线索的嗅觉检测, 以调解其溯河产卵生命史, 包括三个不同阶段:幼虫, 幼仔和成年。海洋鳗幼虫钻入淡水溪流的沉积物中, 经历剧烈的蜕变, 转化为迁徙到湖泊或海洋的幼鱼, 它们寄生大的寄主鱼类。从宿主鱼分离后, 成虫迁移回产卵流, 由流驻留幼虫15、16、17、18、19 所释放的迁移信息素引导..成熟雄性上升到产卵场, 释放多组分性信息素吸引配偶, 间歇产卵约一周, 然后死15,20。海洋鳗信息素的识别是很重要的, 因为对劳伦琴大湖21的入侵性海洋鳗的控制, 是一种调控费洛蒙传播系统的方法。

<table border="0" cellpadding="0" cellspacing="0" width="1135">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:340px;">Premium standard wall borosilicate capillaries with filament&#160;</td>
			<td style="width:199px;">Warner Instruments</td>
			<td style="width:160px;">G150F-4</td>
			<td style="width:436px;">recording and reference electrode (OD 1.5 mm, ID 0.86 mm)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pipette puller instrument&#160;</td>
			<td style="width:199px;">Narishige</td>
			<td style="width:160px;">PC-10</td>
			<td>pulls electrodes for EOGs</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Diamond-tipped glass cutter</td>
			<td style="width:199px;">Generic</td>
			<td style="width:160px;"></td>
			<td>cut tip of electrodes for EOG</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Borosilicate glass capillaries</td>
			<td style="width:199px;">World Precision Instruments</td>
			<td style="width:160px;">1B150-4</td>
			<td>odorant delivery tube for EOG</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:340px;">Recording electrode holder E Series straight body with Ag/AgCl pellet for glass capillary OD 1.5 mm</td>
			<td style="width:199px;">Warner Instruments</td>
			<td style="width:160px;">ESP-M15N</td>
			<td>recording electrode holder</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:340px;">Reference electrode holder E Series with handle with&#160; Ag/AgCl pellet&#160; for glass capillary OD 1.5 mm</td>
			<td style="width:199px;">Warner Instruments</td>
			<td style="width:160px;">E45P-F15NH</td>
			<td>reference electrode holder</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">1 mm pin</td>
			<td style="width:199px;">Warner Instruments</td>
			<td style="width:160px;">WC1-10</td>
			<td>to bridge reference and recording electrode holders</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">2 mm pin</td>
			<td style="width:199px;">Warner Instruments</td>
			<td style="width:160px;">WC2-5</td>
			<td>to bridge reference and recording electrode holders</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">Agar</td>
			<td style="width:199px;">Sigma</td>
			<td style="width:160px;">A1296</td>
			<td>molten agar to fill electrodes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">Potassium chloride (KCl)</td>
			<td style="width:199px;">Sigma</td>
			<td style="width:160px;">P9333</td>
			<td>3M KCl to fill electrodes and electrode holders</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">Micropipette microfil</td>
			<td style="width:199px;">World Precision Instruments</td>
			<td style="width:160px;">MF28G-5</td>
			<td>to fill electrodes and electrode holders&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">L-Arginine</td>
			<td style="width:199px;">Sigma</td>
			<td style="width:160px;">A5006</td>
			<td>positive control odorant for EOG</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">Methanol</td>
			<td style="width:199px;">Sigma</td>
			<td style="width:160px;">34860</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">Water bath</td>
			<td style="width:199px;">Custom made</td>
			<td style="width:160px;">N/A</td>
			<td>holds odorants for EOG</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">3-aminobenzoic acid ethyl ester (MS222)</td>
			<td style="width:199px;">Syndel USA</td>
			<td>Tricaine1G</td>
			<td>EOG anesthetic&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">Gallamine triethiodide</td>
			<td style="width:199px;">Sigma</td>
			<td style="width:160px;">G8134-5G</td>
			<td>EOG paralytic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">1 mL syringe</td>
			<td style="width:199px;">BD Biosciences</td>
			<td>301025</td>
			<td>to administer paralytic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">Subcutaneous needle 26G 5/8</td>
			<td style="width:199px;">BD Biosciences</td>
			<td style="width:160px;">305115</td>
			<td>to administer paralytic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">Roller clamp</td>
			<td style="width:199px;">World Precision Instruments</td>
			<td style="width:160px;">14043-20</td>
			<td>adjust flow rate of anesthic into lamprey&#39;s mouth</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">Sodium chloride (NaCl)</td>
			<td style="width:199px;">J.T. Baker</td>
			<td style="width:160px;">3624-05</td>
			<td>for preparation of 0.9% saline</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">V-shaped plastic stand as specimen stage</td>
			<td style="width:199px;">Custom made</td>
			<td style="width:160px;">N/A</td>
			<td>holds lamprey during EOG</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">Plastic trough</td>
			<td style="width:199px;">Custom made</td>
			<td style="width:160px;">N/A</td>
			<td>holds V-shaped plastic stand during EOG</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">Scalpel Blades - #11</td>
			<td style="width:199px;">Fine Science Tools</td>
			<td style="width:160px;">10011-00</td>
			<td>for EOG dissection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">Scalpel Handle - #3</td>
			<td style="width:199px;">Fine Science Tools</td>
			<td style="width:160px;">10003-12</td>
			<td>for EOG dissection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">Straight ultra fine forceps</td>
			<td style="width:199px;">Fine Science Tools</td>
			<td style="width:160px;">11252-00</td>
			<td>for EOG dissection, Dumont #5SF Forceps</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">Curved ultra fine forceps</td>
			<td style="width:199px;">Fine Science Tools</td>
			<td>11370-42</td>
			<td>for EOG dissection, Moria MC40B</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">Straight pring Scissors</td>
			<td style="width:199px;">Fine Science Tools</td>
			<td>15003-08</td>
			<td>for EOG dissection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">Stereomicroscope</td>
			<td style="width:199px;">Zeiss</td>
			<td style="width:160px;">Discovery V8</td>
			<td>for EOG dissection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">Illuminator light</td>
			<td style="width:199px;">Zeiss</td>
			<td style="width:160px;">CL 1500 ECO</td>
			<td>for EOG dissection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">Plastic tubing</td>
			<td>Generic</td>
			<td style="width:160px;"></td>
			<td>to connect re-circulating EOG setup and water baths</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">Odorant delivery tubing&#160;</td>
			<td style="width:199px;">Custom made</td>
			<td style="width:160px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">In line filter and gasket set</td>
			<td style="width:199px;">Lee Company</td>
			<td style="width:160px;">TCFA1201035A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">Micromanipulators</td>
			<td style="width:199px;">Narishige</td>
			<td style="width:160px;">MM-3</td>
			<td>to position electrodes and odorant delivery capillary tube</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Magnetic holding devices</td>
			<td style="width:199px;">Kanetec</td>
			<td style="width:160px;">MB-K</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Valve driver</td>
			<td style="width:199px;">Arduino</td>
			<td style="width:160px;">custom made</td>
			<td>to control the opening of the valve for odor stimulation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Electromagnetic valve</td>
			<td style="width:199px;">Lee Company</td>
			<td style="width:160px;">LFAA1201618H</td>
			<td>valve for odor stimulation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">NeuroLog AC/DC amplifier</td>
			<td>Digitimer Ltd.</td>
			<td style="width:160px;">NL106</td>
			<td>to increase the amplitude of the elictrical signal</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">NeuroLog DC pre-amplifier with headstage</td>
			<td>Digitimer Ltd.</td>
			<td>NL102G</td>
			<td>to increase the amplitude of the elictrical signal</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">Low-pass 60 Hz filter</td>
			<td>Digitimer Ltd.</td>
			<td>NL125</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:340px;">Digitizer</td>
			<td>Molecular Devices LLC</td>
			<td style="width:160px;">Axon Digidata 1440A</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:340px;">Dell computer (OptiPlex 745) running Axoscope data acquistion software</td>
			<td style="width:199px;">Molecular Devices LLC</td>
			<td>AxoScope version 10.4&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">Faraday cage</td>
			<td style="width:199px;">Custom made</td>
			<td style="width:160px;">N/A</td>
			<td>Electromagnetic noise shielding</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">Two-choice maze</td>
			<td style="width:199px;">Custom made</td>
			<td style="width:160px;">N/A</td>
			<td>waterproofed marine grade plywood covered with plastic liner</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">Trash pump</td>
			<td style="width:199px;">Honda</td>
			<td>WT30XK4A</td>
			<td>fills maze with water from nearby river</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:340px;">Peristaltic pump with tubing</td>
			<td style="width:199px;">Cole Parmer</td>
			<td style="width:160px;">Masterflex 07557-00</td>
			<td>to adminster odorants in maze</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">Inverter Generator&#160;</td>
			<td style="width:199px;">Honda</td>
			<td style="width:160px;">EU1000i</td>
			<td>powers perstaltic pump</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">Release cage</td>
			<td style="width:199px;">Custom made</td>
			<td style="width:160px;">N/A</td>
			<td>used to acclimate lamprey in the maze</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">Mesh</td>
			<td>Generic</td>
			<td style="width:160px;"></td>
			<td>used to contain the dimensions of the maze and minimize water turbulance with mesh rollers</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">Buckets (5 gallon)</td>
			<td>Generic</td>
			<td style="width:160px;"></td>
			<td>to mix odorants</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">Flow meter</td>
			<td style="width:199px;">Marsh-McBirney</td>
			<td>Flo-Mate 2000</td>
			<td>to measure discharge</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">XAD 7 HP resin</td>
			<td style="width:199px;">Dow chemical</td>
			<td style="width:160px;">37380-43-1</td>
			<td>for extraction of conditioned water&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">Methanol</td>
			<td style="width:199px;">Sigma</td>
			<td style="width:160px;">34860</td>
			<td>for extraction of conditioned water&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Water bath</td>
			<td style="width:199px;">Yamato</td>
			<td style="width:160px;">BM 200</td>
			<td>for extraction of conditioned water&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:340px;">Freeze dryer</td>
			<td style="width:199px;">Labconco</td>
			<td style="width:160px;">CentriVap&#160; Concentrator</td>
			<td>for extraction of conditioned water&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">chloroform</td>
			<td style="width:199px;">Sigma</td>
			<td style="width:160px;">CX1050</td>
			<td>for isolation of fraction pools</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">Silica gel 70-230 mesh</td>
			<td style="width:199px;">Sigma</td>
			<td style="width:160px;">112926-00-8</td>
			<td>for isolation of fraction pools</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">Silica gel 230-400 mesh</td>
			<td style="width:199px;">Sigma</td>
			<td style="width:160px;">112926-00-8</td>
			<td>for isolation of fraction pools</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">Pre-coated silica gel TLC plates</td>
			<td style="width:199px;">Sigma</td>
			<td style="width:160px;">99571</td>
			<td>for isolation of fraction pools</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">anisaldehyde</td>
			<td style="width:199px;">Sigma</td>
			<td style="width:160px;">A88107</td>
			<td>for isolation of fraction pools</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sephadex LH-20</td>
			<td style="width:199px;">GE Healthcare</td>
			<td style="width:160px;">17-0090-01</td>
			<td>for isolation of fraction pools</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Amberlite XAD 7 HP resin</td>
			<td style="width:199px;">Sigma</td>
			<td style="width:160px;">XAD7HP</td>
			<td>for extraction of conditioned water&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">4, 2.5L capacity glass columns</td>
			<td>Ace Glass Inc.</td>
			<td>5820</td>
			<td>for extraction of conditioned water&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Acetone</td>
			<td style="width:199px;">Sigma</td>
			<td style="width:160px;">650501</td>
			<td>for extraction of conditioned water&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TQ-S TOF LC Mass spectrometer (or equivalent)</td>
			<td style="width:199px;">Waters Co.</td>
			<td style="width:160px;">N/A</td>
			<td>for structure elucidation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Binary HPLC pump</td>
			<td style="width:199px;">Waters Co.</td>
			<td style="width:160px;">1525</td>
			<td>for isolation of fraction pools/compounds</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Agilent NMR spectrometer, 900MHz (or equivalent)</td>
			<td style="width:199px;">Agilent</td>
			<td style="width:160px;">N/A</td>
			<td>for structure elucidation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Rotovap drying system</td>
			<td style="width:199px;">Buchi</td>
			<td style="width:160px;">RII</td>
			<td>for extraction of conditioned water&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">UV lamp (254 nm)</td>
			<td style="width:199px;">Spectronics Co.</td>
			<td style="width:160px;">ENF-240C</td>
			<td>for thin layer chromatography&#160;</td>
		</tr>
	</tbody>
</table>

the identification of sea lamprey pheromones using bioassay-guided fractionation

生物测定引导分馏是利用生理和行为生物鉴定的结果来指导活性信息素化合物的分离和鉴定的一种迭代方法。这种方法的成功表征了化学信号的功能作为信息素在广泛的动物物种。海洋鳗依赖嗅觉来检测介导行为或生理反应的费洛蒙。我们利用这一知识的鱼类生物学的假设的功能, 假设的信息素, 并指导的分离和鉴定活性的费洛蒙成分。色谱用于从条件水中提取、浓缩和分离化合物。电 olfactogram (EOG) 记录, 以确定哪些分数引出嗅觉反应。两个选择迷宫行为化验, 然后用来确定是否有任何气味的分数也行为上活跃和诱发偏好。光谱和光谱方法提供了分子量和结构信息, 以协助结构的澄清。通过 EOG 和行为学测定, 证实了纯化合物的生物活性。在迷宫中观察到的行为响应最终应在字段设置中进行验证, 以确认它们在自然流设置中的作用。这些生物鉴定发挥双重作用到 1) 指导分馏过程和 2) 确认并进一步确定分离组分的活性。在这里, 我们报告了一个海洋鳗信息素鉴定的代表性结果, 证明了生物测定指导分馏方法的效用。海洋鳗信息素的识别是特别重要的, 因为它是控制劳伦琴大湖侵入性海鳗的选择之一。该方法可以很容易地适应于广泛的分类群中的化学通信, 并揭示了水化学生态学的特点。

图 1中概述了生物测定导向分馏的协议中描述的步骤。该协议涉及的步骤, 以分离和特征的结构, 嗅觉能力和行为活动的5假定海鳗信息素 (图 2)。利用质谱和核磁共振数据 (图 3和图 4), 从条件与成熟的雄性海鳗22、29的水中阐明了 petromyzene a-B 和 petromyzone a C 的结构。
我们的 EOG 记录的代表数据 (图 5) 表明, petromyzene a B 和 petromyzone a C 是所有有力的气味, 刺激成人海鳗嗅上皮和低检测阈值 (图 6).特别值得注意的是, EOG 录音需要适当地放置在嗅觉上皮表面相对于刺激传递管的记录电极, 从而产生良好的信噪比, 可靠的气味响应记录 (图 5B)。如果不遵循这一关键步骤, 就很难识别气味在高噪声中引起的信号。气味暴露后 EOG 迹的向下偏转是一个负电位。一个好的 EOG 信号应该显示一个气味的管理, 导致快速和尖锐的反应, 然后恢复到基线。对所假定的信息素的嗅觉反应也被规范化为 10-5 M l-精氨酸的反应, 一个积极的控制气味在海鳗, 测试整个实验, 以确保记录的完整性保持。
在二选择迷宫行为分析 (图 7A), 排卵女性海鳗被吸引了 petromyzone a, petromyzene a, 并且 petromyzene b, 并且击退从 petromyzone c 排卵女性似乎被击退从 petromyzone B;然而, 行为反应并不显著 (图 7B)。由于 petromyzone B 的数量有限, 因此不可能有较大的样本量。为了正确评估气味的行为响应, 记录每个鳗的控制和实验通道的预处理偏差是至关重要的。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58059/58059fig1.jpg"> 图 1: 以生物测定为导向的费洛蒙分馏的流程图.这个数字是从李, Buchinger 和李30的图2改编的。这些盒子表明了从椭圆形中标明的技术产生的化学产物。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58059/58059fig1large.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/58059/58059fig2.jpg"> 图 2: petromyzene a B 和 petromyzone 的化学结构.这个数字是从图1在李等的修改。22,29.<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58059/58059fig2large.jpg" target="_blank">请点击这里查看这个数字的更大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58059/58059fig3.jpg"> 图 3: 1D 核磁共振谱.1D. petromyzene 的核磁共振谱: (a) 1H 核磁共振 (900 兆赫) 谱和 (B) 13C nmr (225 兆赫) 光谱。这个数字是来自李等的支持信息。29.<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58059/58059fig3large.jpg" target="_blank">请点击这里查看这个数字的更大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/58059/58059fig4.jpg"> 图 4: 2D 核磁共振谱.2D. petromyzene 的核磁共振谱: (a) HSQC 谱, (B) 1h-1h 舒适谱, (C) HMBC 谱, (D) NOESY 谱。这个数字是来自李等的支持信息。29.<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58059/58059fig4large.jpg" target="_blank">请点击这里查看这个数字的更大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/58059/58059fig5.jpg"> 图 5: 电 olfactogram (EOG) 记录准备和代表跟踪记录.(A) 这是一个 EOG 的准备, 显示暴露的海鳗嗅上皮与记录电极, 参考电极, 气味输送管, 和含氧水与麻醉。(B) 本小组显示有代表性的跟踪记录, 表明良好 (顶部) 或坏 (底部) 信噪比。可靠的气味响应记录需要良好的信噪比。气味暴露后 EOG 迹的向下偏转是一个负电位。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58059/58059fig5large.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/58059/58059fig6.jpg"> 图 6: 电 olfactogram (EOG) 浓度-响应曲线的半对数图.Petromyzone 和 petromyzene a 和 B 对成人海水鳗嗅上皮刺激, 检测阈值低。将数据作为均值归一化 EOG 振幅给出 S.E.M。样本大小如下: petromyzone a、petromyzene a、petromyzene b (n = 7) 和 petromyzone b 和 petromyzone C (n = 5)。嵌入是 EOG 响应的扩展视图, 显示响应阈值浓度。这一数字已从李等图3中修改。22和图4在李等。29.<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58059/58059fig6large.jpg" target="_blank">请点击这里查看这个数字的更大版本.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/58059/58059fig7.jpg"> 图 7: 两个选择迷宫的示意图和结果, 用于评估排卵女性海鳗对气味的行为反应.(A) 箭头代表水流的方向 (0.07 米 s-1 0.01)。圈子代表气味管理点。小虚线代表细网格, 用来限制海洋鳗到迷宫的范围内的运动。大虚线表示用于减少水湍流的流动板。灰色矩形表示释放保持架。刻度条 = 1 m。这个数字是从图 S1 在李等。29. (b) 妇女被吸引到 petromyzone a、petromyzene a 和 petromyzene B。在气味曝光之前和之后鳗在迷宫的每个通道上花费的时间 (10-12米) 被用来计算一个偏好指数来评估它对气味的行为反应。偏好指数的正值表示吸引力。样本大小n, 在括号外报告, 括号中的数字表示在实验频道中, 与对照组相比, 在试验通道中花费更多时间的女性人数。数据显示为平均值 S.E.M. (* p < 0.05;魏氏签名等级测试) 比较气味暴露前后的行为偏好。这一数字已从李等图4中修改。22和图5在李等。29.<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58059/58059fig7large.jpg" target="_blank">请点击这里查看这个数字的更大版本.</a>
补充图 1: 迷宫的照片.<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58059/Supplemental_file_maze.tif">请单击此处下载此文件.</a>

Watch this Scientific Journal Video about The Identification of Sea Lamprey Pheromones Using Bioassay-Guided Fractionation at JoVE.com

The Identification of Sea Lamprey Pheromones Using Bioassay-Guided Fractionation

在这里, 我们提出了一个协议, 以分离和描述的结构, 嗅觉效力, 和行为反应的推测信息素化合物的海洋鳗。

利用生物测定引导分馏法鉴定海洋鳗信息素

Bioassay-guided fractionation is an iterative approach that uses the results of physiological and behavioral bioassays to guide the isolation and ...

the-identification-sea-lamprey-pheromones-using-bioassay-guided

Nanyang Technological University

Research

JoVE Journal

Biochemistry

8.9K 次观看.  Michigan State University. 在这里, 我们提出了一个协议, 以分离和描述的结构, 嗅觉效力, 和行为反应的推测信息素化合物的海洋鳗。

视频: The Identification of Sea Lamprey Pheromones Using Bioassay-Guided Fractionation

Identification of Fatty Acids in Bacillus cereus

The Bacillus species contain branched chain and unsaturated fatty acids (FAs) with diverse positions of the methyl branch (iso or anteiso) and of the double bond. Changes in FA composition play a crucial role in the adaptation of bacteria to their environment. These modifications entail a change in the ratio of iso versus anteiso branched FAs, and in the proportion of unsaturated FAs relative to saturated FAs, with double bonds created at specific positions. Precise identification of the FA profile is necessary to understand the adaptation mechanisms of Bacillus species.
Many of the FAs from Bacillus are not commercially available. The strategy proposed herein identifies FAs by combining information on the retention time (by calculation of the equivalent chain length (ECL)) with the mass spectra of three types of FA derivatives: fatty acid methyl esters (FAMEs), 4,4-dimethyl oxazoline derivatives (DMOX), and 3-pyridylcarbinyl ester (picolinyl). This method can identify the FAs without the need to purify the unknown FAs.
Comparing chromatographic profiles of FAME prepared from Bacillus cereus with a commercial mixture of standards allows for the identification of straight-chain saturated FAs, the calculation of the ECL, and hypotheses on the identity of the other FAs. FAMEs of branched saturated FAs, iso or anteiso, display a constant negative shift in the ECL, compared to linear saturated FAs with the same number of carbons. FAMEs of unsaturated FAs can be detected by the mass of their molecular ions, and result in a positive shift in the ECL compared to the corresponding saturated FAs.
The branching position of FAs and the double bond position of unsaturated FAs can be identified by the electron ionization mass spectra of picolinyl and DMOX derivatives, respectively. This approach identifies all the unknown saturated branched FAs, unsaturated straight-chain FAs and unsaturated branched FAs from the B. cereus extract.

Identification of Fatty Acids in Bacillus cereus

We propose a protocol to identify fatty acids without the need to purify them. It combines information on the retention times with the mass spectra of three types of fatty acid derivatives: fatty acid methyl esters (FAMEs), 4,4-dimethyl oxazoline derivatives (DMOX), and 3-pyridylcarbinyl esters (picolinyl).

脂肪酸鉴定<em&gt;蜡状芽孢杆菌</em

The Bacillus species contain branched chain and unsaturated fatty acids (FAs) with diverse positions of the methyl branch (iso or anteiso) and of the ...

科研

生物化学

Nuclear Magnetic Resonance Spectroscopy for the Identification of Multiple Phosphorylations of Intrinsically Disordered Proteins

Aggregates of the neuronal Tau protein are found inside neurons of Alzheimer's disease patients. Development of the disease is accompanied by increased, abnormal phosphorylation of Tau. In the course of the molecular investigation of Tau functions and dysfunctions in the disease, nuclear magnetic resonance (NMR) spectroscopy is used to identify the multiple phosphorylations of Tau. We present here detailed protocols of recombinant production of Tau in bacteria, with isotopic enrichment for NMR studies. Purification steps that take advantage of Tau's heat stability and high isoelectric point are described. The protocol for in vitro phosphorylation of Tau by recombinant activated ERK2 allows for generating multiple phosphorylations. The protein sample is ready for data acquisition at the issue of these steps. The parameter setup to start recording on the spectrometer is considered next. Finally, the strategy to identify phosphorylation sites of modified Tau, based on NMR data, is explained. The benefit of this methodology compared to other techniques used to identify phosphorylation sites, such as immuno-detection or mass spectrometry (MS), is discussed.

We describe here a method to identify multiple phosphorylations of an intrinsically disordered protein by Nuclear Magnetic Resonance Spectroscopy (NMR), using Tau protein as a case study. Recombinant Tau is isotopically enriched and modified in vitro by a kinase prior to data acquisition and analysis.

核磁共振光谱法本质无序蛋白的磷酸化多元的鉴定

Aggregates of the neuronal Tau protein are found inside neurons of Alzheimer's disease patients. Development of the disease is accompanied by increased, ...

A Protein Preparation Method for the High-throughput Identification of Proteins Interacting with a Nuclear Cofactor Using LC-MS/MS Analysis

Transcriptional coregulators are vital to the efficient transcriptional regulation of nuclear chromatin structure. Coregulators play a variety of roles in regulating transcription. These include the direct interaction with transcription factors, the covalent modification of histones and other proteins, and the occasional chromatin conformation alteration. Accordingly, establishing relatively quick methods for identifying proteins that interact within this network is crucial to enhancing our understanding of the underlying regulatory mechanisms. LC-MS/MS-mediated protein binding partner identification is a validated technique used to analyze protein-protein interactions. By immunoprecipitating a previously-identified member of a protein complex with an antibody (occasionally with an antibody for a tagged protein), it is possible to identify its unknown protein interactions via mass spectrometry analysis. Here, we present a method of protein preparation for the LC-MS/MS-mediated high-throughput identification of protein interactions involving nuclear cofactors and their binding partners. This method allows for a better understanding of the transcriptional regulatory mechanisms of the targeted nuclear factors.

We have established a method for the purification of coregulatory interaction proteins using the LC-MS/MS system.

A蛋白制备方法在蛋白质与核余子交互使用LC-MS / MS分析的高通量鉴定

Transcriptional coregulators are vital to the efficient transcriptional regulation of nuclear chromatin structure. Coregulators play a variety of roles in ...

Fractionation for Resolution of Soluble and Insoluble Huntingtin Species

The accumulation of misfolded proteins is central to pathology in Huntington's disease (HD) and many other neurodegenerative disorders. Specifically, a key pathological feature of HD is the aberrant accumulation of mutant HTT (mHTT) protein into high molecular weight complexes and intracellular inclusion bodies composed of fragments and other proteins. Conventional methods to measure and understand the contributions of various forms of mHTT-containing aggregates include fluorescence microscopy, western blot analysis, and filter trap assays. 
However, most of these methods are conformation specific, and therefore may not resolve the full state of mHTT protein flux due to the complex nature of aggregate solubility and resolution.
For the identification of aggregated mHTT and various modified forms and complexes, separation and solubilization of the cellular aggregates and fragments is mandatory. Here we describe a method to isolate and visualize soluble mHTT, monomers, oligomers, fragments, and an insoluble high molecular weight (HMW) accumulated mHTT species. HMW mHTT tracks with disease progression, corresponds with mouse behavior readouts, and has been beneficially modulated by certain therapeutic interventions1. This approach can be used with mouse brain, peripheral tissues, and cell culture but may be adapted to other model systems or disease contexts.

A method is described for fractionation of insoluble and soluble mutant huntingtin species from mouse brain and cell culture. The method described is useful for characterization and quantification of huntingtin protein flux and aids in analyzing protein homeostasis in disease pathogenesis and in the presence of perturbations

可溶性和不溶性杭丁顿蛋白的分离分解

The accumulation of misfolded proteins is central to pathology in Huntington's disease (HD) and many other neurodegenerative disorders. Specifically, a ...

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

The Rho GTPase family belongs to the Ras superfamily and includes approximately 20 members in humans. Rho GTPases are important in the regulation of diverse cellular functions, including cytoskeletal dynamics, cell motility, cell polarity, axonal guidance, vesicular trafficking, and cell cycle control. Changes in Rho GTPase signaling play an essential regulatory role in many pathological conditions, such as&#160;cancer, central nervous system diseases, and immune system-dependent diseases. The posttranslational modification of Rho GTPases (i.e., prenylation by mevalonate pathway intermediates) and GTP binding are key factors which affect the activation of this protein. In this paper, two essential and simple methods are provided to detect a broad range of Rho GTPase prenylation and GTP binding activities. Details of the technical procedures that have been used are explained step by step in this manuscript.

Here we describe a protocol to investigate the prenylation and guanosine-5'-triphosphate (GTP)-loading of Rho GTPase. This protocol consists of two detailed methods, namely membrane fractionation and a GTPase-linked immunosorbent assay. The protocol can be used for measuring the prenylation and GTP loading of different other small GTPases.

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

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

Cell Fractionation of U937 Cells in the Absence of High-speed Centrifugation

In this protocol we detail a method to obtain subcellular fractions of U937 cells without the use of ultracentrifugation or indiscriminate detergents. This method utilizes hypotonic buffers, digitonin, mechanical lysis and differential centrifugation to isolate the cytoplasm, mitochondria and plasma membrane. The process can be scaled to accommodate the needs of researchers, is inexpensive and straightforward. This method will allow researchers to determine protein localization in cells without specialized centrifuges and without the use of commercial kits, both of which can be prohibitively expensive. We have successfully used this method to separate cytosolic, plasma membrane and mitochondrial proteins in the human monocyte cell line U937.

Here, we present a protocol to isolate the plasma membrane, cytoplasm and mitochondria of U937 cells without the use of high-speed centrifugation. This technique can be used to purify subcellular fractions for subsequent examination of protein localization via immunoblotting.

高速离心条件下 u937 细胞的细胞分裂

In this protocol we detail a method to obtain subcellular fractions of U937 cells without the use of ultracentrifugation or indiscriminate detergents. ...

Identification of Novel CK2 Kinase Substrates Using a Versatile Biochemical Approach

The study of kinase-substrate relationships is essential to gain a complete understanding of the functions of these enzymes and their downstream targets in both physiological and pathological states. CK2 is an evolutionarily conserved serine/threonine kinase with a growing list of hundreds of substrates involved in multiple cellular processes. Due to its pleiotropic properties, identifying and characterizing a comprehensive set of CK2 substrates has been particularly challenging and remains a hurdle in the study of this important enzyme. To address this challenge, we have devised a versatile experimental strategy that enables the targeted enrichment and identification of putative CK2 substrates. This protocol takes advantage of the unique dual co-substrate specificity of CK2 allowing for specific thiophosphorylation of its substrates in a cell or tissue lysate. These substrate proteins are subsequently alkylated, immunoprecipitated, and identified by liquid chromatography/tandem mass spectrometry (LC-MS/MS). We have previously used this approach to successfully identify CK2 substrates from Drosophila ovaries and here we extend the application of this protocol to human glioblastoma cells, illustrating the adaptability of this method to investigate the biological roles of this kinase in various model organisms and experimental systems.

The objective of this protocol is to label, enrich, and identify substrates of protein kinase CK2 from a complex biological sample such as a cell lysate or tissue homogenate. This method leverages unique aspects of CK2 biology for this purpose.

利用多功能生化方法鉴定新型 ck2 激酶基材

The study of kinase-substrate relationships is essential to gain a complete understanding of the functions of these enzymes and their downstream targets ...

Identification of Orexin and Endocannabinoid Receptors in Adult Zebrafish Using Immunoperoxidase and Immunofluorescence Methods

Immunohistochemistry (IHC) is a highly sensitive and specific technique involved in the detection of target antigens in tissue sections with labeled antibodies. It is a multistep process in which the optimization of each step is crucial to obtain the optimum specific signal. Through IHC, the distribution and localization of specific biomarkers can be detected, revealing information on evolutionary conservation. Moreover, IHC allows for the understanding of expression and distribution changes of biomarkers in pathological conditions, such as obesity. IHC, mainly the immunofluorescence technique, can be used in adult zebrafish to detect the organization and distribution of phylogenetically conserved molecules, but a standard IHC protocol is not estasblished. Orexin and endocannabinoid are two highly conserved systems involved in the control of food intake and obesity pathology. Reported here are protocols used to obtain information about orexin peptide (OXA), orexin receptor (OX-2R), and cannabinoid receptor (CB1R) localization and distribution in the gut and brain of normal and diet-induced obese (DIO) adult zebrafish models. Also described are methods for immunoperoxidase and double immunofluorescence, as well as preparation of reagents, fixation, paraffin-embedding, and cryoprotection of zebrafish tissue and preparation for an endogenous activity-blocking step and background counterstaining. The complete set of parameters is obtained from previous IHC experiments, through which we have shown how immunofluorescence can help with the understanding of OXs, OX-2R, and CB1R distribution, localization, and conservation of expression in adult zebrafish tissues. The resulting images with highly specific signal intensity led to the confirmation that zebrafish are suitable animal models for immunohistochemical studies of distribution, localization, and evolutionary conservation of specific biomarkers in physiological and pathological conditions. The protocols presented here are recommended for IHC experiments in adult zebrafish.

Presented here are protocols for immunohistochemical characterization and localization of orexin peptide, orexin receptors, and endocannabinoid receptors in the gut and brains of normal and diet-induced obesity (DIO) adult zebrafish models using immunoperixidase and double immunofluorescence methods.

使用免疫过氧化物酶和免疫荧光方法鉴定成年斑马鱼中的奥列辛和内他大麻素受体

Immunohistochemistry (IHC) is a highly sensitive and specific technique involved in the detection of target antigens in tissue sections with labeled ...

Measuring Interactions between Fluorescent Probes and Lignin in Plant Sections by sFLIM Based on Native Autofluorescence

In lignocellulosic biomass (LB), the activity of enzymes is limited by the appearance of non-specific interactions with lignin during the hydrolysis process, which maintains enzymes far from their substrate. Characterization of these complex interactions is thus a challenge in complex substrates such as LB. The method here measures molecular interactions between fluorophore-tagged molecules and native autofluorescent lignin, to be revealed by F&#246;rster resonance energy transfer (FRET). Contrary to FRET measurements in living cells using two exogenous fluorophores, FRET measurements in plants using lignin is not trivial due to its complex autofluorescence. We have developed an original acquisition and analysis pipeline with correlated observation of two complementary properties of fluorescence: fluorescence emission and lifetime. sFLIM (spectral and fluorescent lifetime imaging microscopy) provides the quantification of these interactions with high sensitivity, revealing different interaction levels between biomolecules and lignin.

This protocol describes an original setup combining spectral and fluorescence lifetime measurements to evaluate F&#246;rster resonance energy transfer (FRET) between rhodamine-based fluorescent probes and lignin polymer directly in thick plant sections.

基于原生自荧光的sFLIM测量植物部分荧光探针和木质素之间的相互作用

In lignocellulosic biomass (LB), the activity of enzymes is limited by the appearance of non-specific interactions with lignin during the hydrolysis ...

Cell Fractionation of U937 Cells by Isopycnic Density Gradient Purification

This protocol describes a method to obtain subcellular protein fractions from mammalian cells using a combination of detergents, mechanical lysis, and isopycnic density gradient centrifugation. The major advantage of this procedure is that it does not rely on the sole use of solubilizing detergents to obtain subcellular fractions. This makes it possible to separate the plasma membrane from other membrane-bound organelles of the cell. This procedure will facilitate the determination of protein localization in cells with a reproducible, scalable, and selective method. This method has been successfully used to isolate cytosolic, nuclear, mitochondrial, and plasma membrane proteins from the human monocyte cell line, U937. Although optimized for this cell line, this procedure may serve as a suitable starting point for the subcellular fractionation of other cell lines. Potential pitfalls of the procedure and how to avoid them are discussed as are alterations that may need to be considered for other cell lines.

This fractionation protocol will allow researchers to isolate cytoplasmic, nuclear, mitochondrial, and membrane proteins from mammalian cells. The latter two subcellular fractions are further purified via isopycnic density gradient.

通过等密度梯度纯化对U937细胞进行细胞分级分离

This protocol describes a method to obtain subcellular protein fractions from mammalian cells using a combination of detergents, mechanical lysis, and ...