这项工作是由美国国家科学基金会资助IOS-0817658的支持。

在此协议的斑马鱼程序已经批准的机构动物护理和使用委员会（IACUC）在弗吉尼亚联邦大学。 1.试剂的制备<ol><li> 4％PFA / PBS中。在通风柜权衡，8g仲甲醛（PFA）。同时仍然在通风柜，溶解干燥的PFA在〜80毫升蒸馏H 2ö搅拌并加热至50℃。边搅拌，加3 - 新鲜的1N NaOH 10滴，直到PFA完全溶解，溶液澄清。从火上移开，并加入100 ml 2倍磷酸盐缓冲液（PBS）。带来体积至200毫升蒸馏水H 2 O冷却至室温，并在使用前确认的7.4 pH值。存放在4℃的暗，但用一个星期内。 </li><li>用100％的甲醇，没有任何补充剂。使用95％乙醇而没有任何补充剂。 </li><li>制备磷酸盐缓冲吐温（PBT）。补充磷酸盐缓冲盐水（PBS）的0.1％吐温-20。添加0。5mL的20％吐温-20原液至100毫升1×PBS中。存放在室温下。 </li><li>制备磷酸盐缓冲的Triton-X（PBTx）。补充的PBS含有0.1％的Triton X-100。加入0.5毫升20％的Triton X-100的股票，以100毫升的PBS。存放在室温下。 </li><li>准备10％NGS / PBTx。正常山羊血清（NGS）嵌段的非特异性结合位点。商店NGS中在-20℃等分试样。使用，将1ml加入9mL的PBTx。 </li><li>制备50％甘油/ PBS中。混合2×PBS中，100％的甘油，完全相等的部分。存放在室温下。 </li></ol> 2.胚胎固定<ol><li> 。（ 如 β-actin的:CAAX-GFP） 育种获得野生型（AB和WIK）或转基因胚胎通过自然交配。如果需要的话，注射用构建体或吗啉代胚胎，如所描述。15,16 </li><li> 提高胚胎。收集胚胎，并如所述在28.5℃下在0.003％1-苯基-2-硫脲（PTU）阻止色素沉着的存在下孵育17 </su<html> p&gt; </li><li> 修正。当胚胎已达到所希望的发育阶段，18个， &#160;麻醉用MESAB胚胎，删除尽可能多的系统中的水可能和补充新鲜4％PFA / PBS 3 - 在室温4小时。注:在时间少于20小时受精后（HPF），修复了之前dechorionation。在经过20 HPF倍，采用在解剖显微镜下2对细点镊子的前固定dechorionate。 </li><li>简单地通过使胚胎通过重力而不离心沉降变化的解决方案。传输使用宽孔移液管的胚胎，如上所述。在加盖的微量离心管1.5毫升17变化的解决方案。 </li><li> 后固定剂后3 - 4小时，除去PFA / PBS中，用100％甲醇和存储替换在-20℃至少48小时。注:对于最大的P-钙调蛋白激酶-II免疫反应，限制总的甲醇储存时间为一个星期。 </li></ol>"&gt; 3。免疫染色全胚胎<ol><li>将最低10胚胎在上限1.5 ml离心管每个实验条件，并贴上标签。 </li><li>允许胚胎定居。除去并弃去甲醇，并用含有0.5毫升递减的乙醇浓度的渐进洗涤再水化，如在接下来的步骤表示。每一步都是一个5分钟洗具摇摆。 </li><li>这些5溶液逐步再水化:66％乙醇/ 33％PBTx，33％乙醇/ 66％PBTx，100％PBTx，100％PBTx（这是PBTx的第一重复），100％PBTx（这是第二个重复PBTx）。 </li><li>删除最后PBTx洗。将0.5ml 10％NGS / PBTx。在室温下孵育至少1小时与温和的摇摆，然后取出并丢弃的解决方案。 </li><li>通过在10％NGS / PBTx稀释制备一级抗体溶液。如果联合免疫，先用较高的亲和力抗体。在这项研究中，孵育稀释1小鼠抗乙酰化微管蛋白的单克隆抗体:500。对于为例即，如果有10个样品中，结合6微升股票抗体溶液用3毫升10％NGS / PBTx的，然后分发0.3ml的此到每个管中。 </li><li>将0.5ml稀释的初级抗体与每个管浸入所有胚胎 - 0.2添加。在室温下伴随温和的摇摆O / N培养。 </li><li>当天上午，删除和丢弃抗体溶液。将0.5ml 2％NGS / PBTx洗去多余的主要抗体。轻轻摇动5分钟。取下并丢弃的解决方案。重复两次。 </li><li>暗淡顶灯和稀适当荧光标记的二抗在10％NGS / PBTx使得每个条件包含至少0.5毫升用小鼠抗微管蛋白乙酰化的第一抗体，使用绿色荧光染料的山羊抗小鼠IgG在1:500的稀释。 </li><li>孵育次级抗体进行4小时以在室温下在黑暗中轻轻摇动。从现在起，处理样品下变暗的灯光，并在黑暗的容器孵化或在铝箔包装。 </li><li>在温育结束时，除去并丢弃二级抗体溶液。将0.5ml 2％NGS / PBTx洗。轻轻摇动5分钟。取下并丢弃的解决方案。重复两次。 </li><li>如果共免疫染色，来自不同物种比第一初级抗体获得第二初级抗体。在这些研究中，通过红色荧光染料缀合的山羊抗兔IgG二级抗体跟随兔子抗磷酸钙调蛋白激酶-Ⅱ抗体。 </li><li>稀释兔抗磷酸钙调蛋白激酶-II抗体1:20。对于胚胎的各管，暂停0.3含10％NGS / PBTx，再加入15微升股票抗体溶液。 </li><li>确保胚胎都沉浸和灯光会变暗。在黑暗中轻轻摇摆O / N孵育RT。 </li><li>在昏暗的灯光上午，删除和丢弃抗体溶液。加入0.5毫升2-％NGS / PBTx。轻轻摇动5分钟。取下并丢弃的解决方案。重复两次。 </li><li>保持顶灯暗，淡化相应的荧光conju足够门控的第二抗体，使每个条件包含至少0.5毫升在这项研究中，淡化红色荧光染料缀合的山羊抗兔IgG二级抗体（红色通道）1:500在10％NGS / PBTx。 </li><li>孵育第二次级抗体进行4小时以在室温下在黑暗中轻轻摇动。 </li><li>在此孵化，并在昏暗的灯光年底，捞出弃去二抗溶液。将0.5ml PBTx。轻轻摇动5分钟。取下并丢弃的解决方案。重复两次。商店在任PBTx或50％甘油/ PBS中根据成像过程。 </li></ol> 4.共焦成像和处理<ol><li>摩胚胎成像。将1 - 5胚胎在玻片上。创建主盖玻片并用在腔室的每一侧盖玻片片段滑动之间的腔室。通常，四＃1盖玻片被用来制造这些垫片堆栈而不封口。 注:没有安装介质是必要的。 </li><li>使用100X油浸客观上使单个胚胎成焦点采用透射光。关闭灯光。打开共聚焦显微镜。确保适当的激光器（绿色和/或红色）开启和远程对焦附带从事。 </li><li>打开共聚焦程序。在"采集栏，"选择合适的目标。在"XY基本"栏，点击1,024按钮设置的图像大小为1024。 </li><li>在"激光器和探测器"栏，点击红色框488和568的绿色盒子打开激光器和探测器每个通道。针孔设置为中，并调整每个通道以可视化的"增益"栏的增益。 </li><li>在"获取设置"栏中，单击"活着"开始采集图像。在"视图设置"栏中，取消选中"强制积分变焦"框中心"实时"窗口。 </li><li>在"获取设置"栏中的"Z"选项卡下，逐步完成图像的图层。选择后，NU光学切片的MBER掺入到图象，将移动到某一点在z平面的"中心"。 </li><li>在"Z"选项卡，单击红色的小"引用"对话框。这将归零RFA在选定为"中心"的地步。在标有"步长"的方框中，选择图层（0.25和2.0微米之间微米是理想的）厚度。需要注意的是"文件大小"框，并试图限制到1GB。 注意:通常情况下，可达0.5 40光学切片 - 为1.0μm而得到的，但可以根据经验来确定区段的数量。 </li><li>要找到图像的顶部极端，点击旁边的圆圈为"top"，并通过Z轴移动图层。现在点击圆圈旁边的"底"，计算机将图像回层为中心。再次通过层移动到找到图像的底部极端。 </li><li>点击在"获取设置""活"再次框停止激光收购。在此面板中，单击标平均红色框和Z堆栈。这些框变成绿色。 </li><li>在"获取设置"框中，单击"单次"收购整个系列的图像。因为它在两个信道，并通过在整个Z堆叠扫描可以监视进展情况。 </li><li>若要保存，单击该卷窗口中，点击"另存为"，并命名该文件。另存为".ids"文件。体积渲染文件，而Z堆栈仍然是开放的，选择数据下拉菜单，点击"批量渲染"。保存文件渲染为TIFF文件。这是本出版物中显示的投影图像。 </li></ol>

<ol>
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作者宣称，他们没有竞争的经济利益。

煤灰/甲醇方法，在这个实验室使用斑马鱼发育期间优化磷酸-T 287 -CaMK-II型表位的免疫定位的主要目的开发的。这种方法的几个纤毛器官，包括斑马鱼KV，12内耳14和肾脏。13特别是在对KV阶段，这种技术是必要的形成过程中成功地本地化的P-钙调蛋白激酶-II该方法的成功可能是由于一个）的自体荧光的最小化，b）该磷钙调蛋白激酶-II型表位的保存和c）的表位的抗体?...

The techniques described here are the outcome of studies that have sought to define downstream targets of Ca2+ signals during events that occur during early development, including fertilization, gastrulation, somitogenesis and trunk, eye, brain and organ formation.1-3 The original discoveries of embryonic Ca2+ signaling were dependent on the use of natural and engineered Ca2+ indicators, such as aequorin4 and fura-2.5 Even with current technology, the detection of transient elevations of Ca2+ requires cumbersome analytical tools and does not reveal the targets of such Ca2+ signals. 
This laboratory investigates Ca2+ signals that act through the Ca2+/calmodulin-dependent (multifunctional) protein kinase known as CaMK-II, an enzyme that is enriched in the central nervous system and originally identified as a regulator of long-term potentiation.6 CaMK-II is not brain-specific, is widely expressed and highly conserved throughout the entire lifespan and bodies of species throughout the animal kingdom, including invertebrates.7,8 CaMK-II has the unique capability of sustaining its own activity even after Ca2+ levels have diminished due to its ability to autophosphorylate at Thr287. In this autophosphorylated state, CaMK-II remains active in a Ca2+/CaM-independent manner, until dephosphorylated.6 Thus, the localization of phosphorylated CaMK-II (Thr287) can identify cells in which natural, relevant Ca2+ elevations have occurred. 
An antibody against autophosphorylated (P-Thr287) mammalian CaMK-II has been well-characterized and was initially used to localize activated CaMK-II in brain tissue.9 Zebrafish (Danio rerio) have seven CaMK-II genes10,11 whose protein products contain a sequence of MHRQE[pT287]VECLK in this region.10,11 This sequence is very similar to the phosphopeptide antigen used to create this rabbit polyclonal antibody (MHRQE[pT]VDCLK; Upstate/Millipore) and therefore it was not a complete surprise that this antibody cross-reacted with zebrafish CaMK-II. This laboratory showed that this antibody reacts with zebrafish CaMK-II in proportion to autophosphorylation and Ca2+/CaM-independent activity.12 Additional pan-specific CaMK-II antibodies have also been shown to cross-react with zebrafish CaMK-II.13
This antibody has been used to demonstrate that zebrafish CaMK-II is preferentially activated in cells on one side of the zebrafish Kupffer's Vesicle (KV), the ciliated organ necessary for establishment of left/right asymmetry.12 This antibody was used to demonstrate that CaMK-II is transiently activated in four adjacent cells on the left side of the KV during the exact same developmental phase that organ positioning is determined.12 In addition to the Kupffer's Vesicle (KV), autophosphorylated (P-T287) was also located in specific intracellular sites in other ciliated tissues including the kidney, neuromasts, and inner ear.12,13 In the zebrafish kidney, P-T287-CaMK-II is enriched along the apical border of ciliated ductal cells and within cloacal cilia where it influences their assembly.13 Finally, in the developing inner ear, P-T287-CaMK-II is intensely concentrated at the base of cilia and influences cell differentiation through the Delta-Notch signal pathway.14 In summary, the detection of activated CaMK-II has pinpointed sites of intracellular Ca2+ release and illuminated potential new signaling pathways.
These discoveries were completely dependent on developing a sensitive and accurate method to localize activated (P-T287-autophosphorylated) CaMK-II. The methods to fix and immunostain the zebrafish KV, kidney and inner ear are described. The limitations of this technique are also described. These techniques should be useful to any investigator who seeks to obtain high-resolution images in two fluorescent channels of not just phospho-epitopes, but any epitope, during early vertebrate development.

<table border="0" cellpadding="0" cellspacing="0" width="886">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">1-phenyl-2-thiourea (PTU)</td>
			<td style="width:175px;">Sigma</td>
			<td style="width:119px;">P-7629</td>
			<td style="width:347px;">0.12% Stock solution. Dilute 1:40 in system water</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Alexa488 anti-mouse IgG</td>
			<td style="width:175px;">Life Technologies</td>
			<td style="width:119px;">A11001</td>
			<td style="width:347px;">Goat polyclonal, use at 1:500</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Alexa488 anti-rabbit IgG</td>
			<td style="width:175px;">Life Technologies</td>
			<td style="width:119px;">A11008</td>
			<td style="width:347px;">Goat polyclonal, use at 1:500</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Alexa488 phalloidin</td>
			<td style="width:175px;">Life Technologies</td>
			<td style="width:119px;">A12379</td>
			<td>Preferentially binds to F-actin</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Alexa568 anti-mouse IgG</td>
			<td style="width:175px;">Life Technologies</td>
			<td style="width:119px;">A11004</td>
			<td style="width:347px;">Goat polyclonal, use at 1:500</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Alexa568 anti-rabbit IgG</td>
			<td style="width:175px;">Life Technologies</td>
			<td style="width:119px;">A11011</td>
			<td style="width:347px;">Goat polyclonal, use at 1:500</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">anti-acetylated a-tubulin</td>
			<td style="width:175px;">Sigma</td>
			<td style="width:119px;">T7451</td>
			<td style="width:347px;">Mouse monoclonal, use at 1:500</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:247px;">anti-phospho-T287 CaMK-II</td>
			<td style="width:175px;">EMD Millipore</td>
			<td style="width:119px;">06-881</td>
			<td style="width:347px;">Rabbit polyclonal, use at 1:20</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">anti-total CaMK-II</td>
			<td style="width:175px;">BD Biosciences</td>
			<td style="width:119px;">611292</td>
			<td style="width:347px;">Mouse monoclonal, use at 1:20</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Ethanol</td>
			<td style="width:175px;">Fisher</td>
			<td style="width:119px;">S96857</td>
			<td style="width:347px;">Lab grade, 95% denatured</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Forceps</td>
			<td style="width:175px;">Fine Science Tools</td>
			<td>11252-20</td>
			<td>Dumont #5</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Glass coverslips</td>
			<td style="width:175px;">VWR</td>
			<td style="width:119px;">16004-330</td>
			<td>#1&#160; thickness</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Glass microscope slides</td>
			<td style="width:175px;">Fisher</td>
			<td style="width:119px;">12-550-15</td>
			<td>Standard glass slides</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Methanol</td>
			<td style="width:175px;">Fisher</td>
			<td style="width:119px;">A411</td>
			<td>Store in freezer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Microcentrifuge tubes</td>
			<td style="width:175px;">VWR</td>
			<td style="width:119px;">20170-038</td>
			<td>capped tubes, not sterile</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Normal goat serum</td>
			<td style="width:175px;">Life Technologies</td>
			<td style="width:119px;">16210-064</td>
			<td>Aliquot 1ml tubes, store in freezer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Paraformaldehyde</td>
			<td style="width:175px;">Sigma</td>
			<td style="width:119px;">P-6148</td>
			<td>Reagent grade, crystalline</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Phosphate buffered saline (PBS)</td>
			<td style="width:175px;">Quality Biological</td>
			<td style="width:119px;">119-069-131</td>
			<td>10X stock solution or made in lab</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Triton X-100</td>
			<td style="width:175px;">Sigma</td>
			<td style="width:119px;">BP-151</td>
			<td>10% solution in water, store at room temp</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Tween-20</td>
			<td style="width:175px;">Life Technologies</td>
			<td style="width:119px;">85113</td>
			<td>10% solution in water, store at room temp</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Compound microscope</td>
			<td style="width:175px;">Nikon</td>
			<td style="width:119px;">E-600</td>
			<td>Mount on vibration-free table</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">C1 Plus two-laser scanning confocal</td>
			<td style="width:175px;">Nikon</td>
			<td style="width:119px;">C1 Plus</td>
			<td>Run by EZ-C1 program, but upgrades use &#34;Elements&#34;</td>
		</tr>
	</tbody>
</table>

immunostaining phospho-epitopes in ciliated organs of whole mount zebrafish embryos

细胞迅速增殖，基因的组织特异性表达和信令网络的出现表征所有脊椎动物的早期胚胎发育。即使在单个细胞 - - 动力学和信号的位置，在胚胎发育补充重要的发育基因的鉴定。免疫染色技术描述已被证明以限定小至初级纤毛结构的细胞内和整个动物的信号的动力学。使用激光扫描共聚焦显微镜复合固定，成像和图像处理技术可以在短短36小时内完成。 斑马鱼（ 斑马鱼 ）是谁寻求脊椎动物物种是负担得起的和相关的人类疾病进行研究调查一个理想的有机体。遗传击倒或击倒，必须通过实际的蛋白质产物的损失来确认。蛋白质丢失这样的确认可以使用这里描述的技术来实现。线索到信号通路，也可以通过使用与已经由磷酸化的翻译后修饰的蛋白反应的抗体破译。维护和优化表位的磷酸化状态，因此这一决定的关键，并通过该协议来实现的。 这项研究的发展和第一72小时期间介绍了技术修复的胚胎共定位的各种与在枯否囊泡（KV）纤毛相关的表位，肾脏和内耳。这些技术是简单的，不需要清扫，并可以在一个相对较短的时间内完成。焦投影图像堆栈成一个单一的形象呈现这些数据的有效手段。

可视化的磷酸化表位条件最优 描述蛋白抗原决定簇在斑马鱼胚胎的免疫组化方法相比，通过原位杂交本地化的mRNAs一直比较稀疏。在定位在斑马鱼胚胎的纤毛细胞蛋白的表位使用的固定剂包括4％PFA / PBS中并登特的固定剂，它是甲醇和DMSO。19定位RNA的由整体原位杂交（WISH）的混合物中，通常?...

Watch this Scientific Journal Video about Immunostaining Phospho-epitopes in Ciliated Organs of Whole Mount Zebrafish Embryos at JoVE.com

Immunostaining Phospho-epitopes in Ciliated Organs of Whole Mount Zebrafish Embryos

技术描述于免疫染色在整个斑马鱼胚胎磷酸的表位，然后进行在小至初级纤毛细胞结构双色荧光共焦定位。用于固定和成像技术可以定义位置和特定蛋白质的外观或活化​​的动力学。

免疫染色磷酸化表位整装斑马鱼胚胎纤毛器官

The rapid proliferation of cells, the tissue-specific expression of genes and the emergence of signaling networks characterize early embryonic development ...

immunostaining-phospho-epitopes-ciliated-organs-whole-mount-zebrafish

Research

JoVE Journal

Chemistry

7.9K 次观看.  Virginia Commonwealth University. 技术描述于免疫染色在整个斑马鱼胚胎磷酸的表位，然后进行在小至初级纤毛细胞结构双色荧光共焦定位。用于固定和成像技术可以定义位置和特定蛋白质的外观或活化​​的动力学。 

视频: Immunostaining Phospho-epitopes in Ciliated Organs of Whole Mount Zebrafish Embryos

High Precision Zinc Isotopic Measurements Applied to Mouse Organs

We present a procedure to measure with high precision zinc isotope ratios in mouse organs. Zinc is composed of 5 stable isotopes (64Zn, 66Zn, 67Zn, 68Zn and 70Zn) which are naturally fractionated between mouse organs. We first show how to dissolve the different organs in order to free the Zn atoms; this step is realized by a mixture of HNO3 and H2O2. We then purify the zinc atoms from all the other elements, in particular from isobaric interferences (e.g., Ni), by anion-exchange chromatography in a dilute HBr/HNO3 medium. These first two steps are performed in a clean laboratory using high purity chemicals. Finally, the isotope ratios are measured by using a multi-collector inductively-coupled-plasma mass-spectrometer, in low resolution. The samples are injected using a spray chamber and the isotopic fractionation induced by the mass-spectrometer is corrected by comparing the ratio of the samples to the ratio of a standard (standard bracketing technique).&#160;This full typical&#160;procedure produces an isotope ratio with a 50 ppm (2 s.d.) reproducibility.

We present the technique to measure with high precision zinc isotope ratios in mouse organs.

适用于鼠标机关高精度锌同位素测量

We present a procedure to measure with high precision zinc isotope ratios in mouse organs. Zinc is composed of 5 stable isotopes (64Zn, 66Zn, 67Zn, 68Zn ...

科研

化学

Radiolabeling and Quantification of Cellular Levels of Phosphoinositides by High Performance Liquid Chromatography-coupled Flow Scintillation

Phosphoinositides (PtdInsPs) are essential signaling lipids responsible for recruiting specific effectors and conferring organelles with molecular identity and function. Each of the seven PtdInsPs varies in their distribution and abundance, which are tightly regulated by specific kinases and phosphatases. The abundance of PtdInsPs can change abruptly in response to various signaling events or disturbance of the regulatory machinery. To understand how these events lead to changes in the amount of PtdInsPs and their resulting impact, it is important to quantify PtdInsP levels before and after a signaling event or between control and abnormal conditions. However, due to their low abundance and similarity, quantifying the relative amounts of each PtdInsP can be challenging. This article describes a method for quantifying PtdInsP levels by metabolically labeling cells with 3H-myo-inositol, which is incorporated into PtdInsPs. Phospholipids are then precipitated and deacylated. The resulting soluble 3H-glycero-inositides are further extracted, separated by high-performance liquid chromatography (HPLC), and detected by flow scintillation. The labeling and processing of yeast samples is described in detail, as well as the instrumental setup for the HPLC and flow scintillator. Despite losing structural information regarding acyl chain content, this method is sensitive and can be optimized to concurrently quantify all seven PtdInsPs in cells.

Phosphoinositides are signaling lipids whose relative abundance rapidly changes in response to various stimuli. This article describes a method to measure the abundance of phosphoinositides by metabolically labeling cells with 3H-myo-inositol, followed by extraction and deacylation. Extracted glycero-inositides are then separated by high-performance liquid chromatography and quantified by flow scintillation.

放射性标记和磷酸肌醇的细胞水平的定量高效液相色谱耦合流动闪烁

Phosphoinositides (PtdInsPs) are essential signaling lipids responsible for recruiting specific effectors and conferring organelles with molecular ...

Sequencing of Plant Wall Heteroxylans Using Enzymic, Chemical (Methylation) and Physical (Mass Spectrometry, Nuclear Magnetic Resonance) Techniques

This protocol describes the specific techniques used for the characterization of reducing end (RE) and internal region glycosyl sequence(s) of heteroxylans. De-starched wheat endosperm cell walls were isolated as an alcohol-insoluble residue (AIR)1 and sequentially extracted with water (W-sol Fr) and 1 M KOH containing 1% NaBH4 (KOH-sol Fr) as described by Ratnayake et al. (2014)2. Two different approaches (see summary in Figure 1) are adopted. In the first, intact W-sol AXs are treated with 2AB to tag the original RE backbone chain sugar residue and then treated with an endoxylanase to generate a mixture of 2AB-labelled RE and internal region reducing oligosaccharides, respectively. In a second approach, the KOH-sol Fr is hydrolyzed with endoxylanase to first generate a mixture of oligosaccharides which are subsequently labelled with 2AB. The enzymically released ((un)tagged) oligosaccharides from both W- and KOH-sol Frs are then methylated and the detailed structural analysis of both the native and methylated oligosaccharides is performed using a combination of MALDI-TOF-MS, RP-HPLC-ESI-QTOF-MS and ESI-MSn. Endoxylanase digested KOH-sol AXs are also characterized by nuclear magnetic resonance (NMR) that also provides information on the anomeric configuration. These techniques can be applied to other classes of polysaccharides using the appropriate endo-hydrolases.

Sequencing of Plant Wall Heteroxylans Using Enzymic, Chemical Methylation and Physical Mass Spectrometry, Nuclear Magnetic Resonance Techniques

This protocol describes the specific techniques used for the structural characterization of reducing end (RE) and internal region glycosyl sequence(s) of heteroxylans by tagging the RE with 2 aminobenzamide prior to enzymatic (endoxylanase) hydrolysis and then analysis of the resultant oligosaccharides using mass spectrometry (MS) and nuclear magnetic resonance (NMR).

使用酶法，化学（甲基化）和物理（质谱，核磁共振）技术，植物墙Heteroxylans测序

This protocol describes the specific techniques used for the characterization of reducing end (RE) and internal region glycosyl sequence(s) of ...

Synthesis of Protein Bioconjugates via Cysteine-maleimide Chemistry

The chemical linking or bioconjugation of proteins to fluorescent dyes, drugs, polymers and other proteins has a broad range of applications, such as the development of antibody drug conjugates (ADCs) and nanomedicine, fluorescent microscopy and systems chemistry. For many of these applications, specificity of the bioconjugation method used is of prime concern. The Michael addition of maleimides with cysteine(s) on the target proteins is highly selective and proceeds rapidly under mild conditions, making it one of the most popular methods for protein bioconjugation.
We demonstrate here the modification of the only surface-accessible cysteine residue on yeast cytochrome c with a ruthenium(II) bisterpyridine maleimide. The protein bioconjugation is verified by gel electrophoresis and purified by aqueous-based fast protein liquid chromatography in 27% yield of isolated protein material. Structural characterization with MALDI-TOF MS and UV-Vis is then used to verify that the bioconjugation is successful. The protocol shown here is easily applicable to other cysteine - maleimide coupling of proteins to other proteins, dyes, drugs or polymers.

Synthesis of Protein Bioconjugates via Cysteine-maleimide Chemistry

This protocol details the important steps required for the bioconjugation of a cysteine containing protein to a maleimide, including reagent purification, reaction conditions, bioconjugate purification and bioconjugate characterization.

蛋白质生物缀合物的合成<em&gt;通过</em&gt;半胱氨酸马来酰亚胺化学

The chemical linking or bioconjugation of proteins to fluorescent dyes, drugs, polymers and other proteins has a broad range of applications, such as the ...

Accessing Valuable Ligand Supports for Transition Metals: A Modified, Intermediate Scale Preparation of 1,2,3,4,5-Pentamethylcyclopentadiene

A reliable, intermediate scale preparation of 1,2,3,4,5-pentamethylcyclopentadiene (Cp*H) is presented, based on modifications of existing protocols that derive from initial 2-bromo-2-butene lithiation followed by acid mediated dienol cyclization. The revised synthesis and purification of the ligand avoids the use of mechanical stirring while still permitting access to significant quantities (39 g) of Cp*H in good yield (58%). The procedure offers other additional benefits, including a more controlled quench of excess lithium during the production of the intermediate heptadienols and a simplified isolation of Cp*H of sufficient purity for metallation with transition metals. The ligand was subsequently used to synthesize [Cp*MCl2]2 complexes of both iridium and ruthenium to demonstrate the utility of the Cp*H prepared and purified by our method. The procedure outlined herein affords substantial quantities of a ubiquitous ancillary ligand support used in organometallic chemistry while minimizing the need for specialized laboratory equipment, thus providing a simpler and more accessible entry point into the chemistry of 1,2,3,4,5-pentamethylcyclopentadiene.

Reliable, intermediate scale preparation of 1,2,3,4,5-pentamethylcyclopentadiene (Cp*H) is presented. The revised protocol for the synthesis and purification of the ligand minimizes the need for specialized laboratory equipment while simplifying reaction workups and product purification. Use of Cp*H in the synthesis of [Cp*MCl2]2 complexes (M = Ru, Ir) is also described.

访问有价值的配体为支持过渡金属:1,2,3,4,5-五甲基环戊二烯的一种改进，中级规模制备

A reliable, intermediate scale preparation of 1,2,3,4,5-pentamethylcyclopentadiene (Cp*H) is presented, based on modifications of existing protocols that ...

Microprobe Capillary Electrophoresis Mass Spectrometry for Single-cell Metabolomics in Live Frog (Xenopus laevis) Embryos

The quantification of small molecules in single cells raises new potentials for better understanding the basic processes that underlie embryonic development. To enable single-cell investigations directly in live embryos, new analytical approaches are needed, particularly those that are sensitive, selective, quantitative, robust, and scalable to different cell sizes. Here, we present a protocol that enables the in situ analysis of metabolism in single cells in freely developing embryos of the South African clawed frog (Xenopus laevis), a powerful model in cell and developmental biology. This approach uses a capillary microprobe to aspirate a defined portion from single identified cells in the embryo, leaving neighboring cells intact for subsequent analysis. The collected cell content is analyzed by a microscale capillary electrophoresis electrospray ionization (CE-ESI) interface coupled to a high-resolution tandem mass spectrometer. This approach is scalable to various cell sizes and compatible with the complex three-dimensional structure of the developing embryo. As an example, we demonstrate that microprobe single-cell CE-ESI-MS enables the elucidation of metabolic cell heterogeneity that unfolds as a progenitor cell gives rise to descendants during development of the embryo. Besides cell and developmental biology, the single-cell analysis protocols described here are amenable to other cell sizes, cell types, or animal models.

Microprobe Capillary Electrophoresis Mass Spectrometry for Single-cell Metabolomics in Live Frog Xenopus laevis Embryos

We describe steps that enable fast in situ sampling of a small portion of an individual cell with high precision and minimal invasion using capillary-based micro-sampling, to facilitate chemical characterization of a snapshot of metabolic activity in live embryos using a custom-built single cell capillary electrophoresis and mass spectrometry platform.

活蛙 (爪蟾) 胚胎单细胞代谢的微探针毛细管电泳质谱

The quantification of small molecules in single cells raises new potentials for better understanding the basic processes that underlie embryonic ...

Photoelectron Imaging of Anions Illustrated by 310 Nm Detachment of F&#8722;

Anion photoelectron imaging is a very efficient method for the study of energy states of bound negative ions, neutral species and interactions of unbound electrons with neutral molecules/atoms. State-of-the-art in vacuo anion generation techniques allow application to a broad range of atomic, molecular, and cluster anion systems. These are separated and selected using time-of-flight mass spectrometry. Electrons are removed by linearly polarized photons (photo detachment) using table-top laser sources which provide ready access to excitation energies from the infra-red to the near ultraviolet. Detecting the photoelectrons with a velocity mapped imaging lens and position sensitive detector means that, in principle, every photoelectron reaches the detector and the detection efficiency is uniform for all kinetic energies. Photoelectron spectra extracted from the images via mathematical reconstruction using an inverse Abel transformation reveal details of the anion internal energy state distribution and the resultant neutral energy states. At low electron kinetic energy, typical resolution is sufficient to reveal energy level differences on the order of a few millielectron-volts, i.e., different vibrational levels for molecular species or spin-orbit splitting in atoms. Photoelectron angular distributions extracted from the inverse Abel transformation represent the signatures of the bound electron orbital, allowing more detailed probing of electronic structure. The spectra and angular distributions also encode details of the interactions between the outgoing electron and the residual neutral species subsequent to excitation. The technique is illustrated by the application to an atomic anion (F&#8722;), but it can also be applied to the measurement of molecular anion spectroscopy, the study of low lying anion resonances (as an alternative to scattering experiments) and femtosecond (fs) time resolved studies of the dynamic evolution of anions.

Here, we present a protocol for photoelectron imaging of anionic species. Anions generated in vacuo and separated by mass spectrometry are probed using velocity mapped photoelectron imaging, providing details of anion and neutral energy levels, anion and neutral structure and the nature of the anion electronic state.

由 F− 310 Nm 支队所示的阴离子光电子成像

Anion photoelectron imaging is a very efficient method for the study of energy states of bound negative ions, neutral species and interactions of unbound ...

Bio-inspired Polydopamine Surface Modification of Nanodiamonds and Its Reduction of Silver Nanoparticles

Surface functionalization of nanodiamonds (NDs) is still challenging due to the diversity of functional groups on the ND surfaces. Here, we demonstrate a simple protocol for the multifunctional surface modification of NDs by using mussel-inspired polydopamine (PDA) coating. In addition, the functional layer of PDA on NDs could serve as a reducing agent to synthesize and stabilize metal nanoparticles. Dopamine (DA) can self-polymerize and spontaneously form PDA layers on ND surfaces if the NDs and dopamine are simply mixed together. The thickness of a PDA layer is controlled by varying the concentration of DA. A typical result shows that a thickness of ~5 to ~15 nm of the PDA layer can be reached by adding 50 to 100 &#181;g/mL of DA to 100 nm ND suspensions. Furthermore, the PDA-NDs are used as a substrate to reduce metal ions, such as Ag[(NH3)2]+, to silver nanoparticles (AgNPs). The sizes of the AgNPs rely on the initial concentrations of Ag[(NH3)2]+. Along with an increase in the concentration of Ag[(NH3)2]+, the number of NPs increases, as well as the diameters of the NPs. In summary, this study not only presents a facile method for modifying the surfaces of NDs with PDA, but also demonstrates the enhanced functionality of NDs by anchoring various species of interest (such as AgNPs) for advanced applications.

A facile protocol is presented to functionalize the surfaces of nanodiamonds with polydopamine.

纳米金刚石的生物激发多巴胺表面改性及其对银纳米粒子的还原

Surface functionalization of nanodiamonds (NDs) is still challenging due to the diversity of functional groups on the ND surfaces. Here, we demonstrate a ...

Synthesis and Bioconjugation of Thiol-Reactive Reagents for the Creation of Site-Selectively Modified Immunoconjugates

Maleimide-bearing bifunctional probes have been employed for decades for the site-selective modification of thiols in biomolecules, especially antibodies. Yet maleimide-based conjugates display limited stability in vivo because the succinimidyl thioether linkage can undergo a retro-Michael reaction. This, of course, can lead to the release of the radioactive payload or its exchange with thiol-bearing biomolecules in circulation. Both of these processes can produce elevated activity concentrations in healthy organs as well as decreased activity concentrations in target tissues, resulting in reduced imaging contrast and lower therapeutic ratios. In 2018, we reported the creation of a modular, stable, and easily accessible phenyloxadiazolyl methyl sulfone reagent &#8212; dubbed &#8216;PODS&#8217; &#8212; as a platform for thiol-based bioconjugations. We have clearly demonstrated that PODS-based site-selective bioconjugations reproducibly and robustly create homogenous, well-defined, highly immunoreactive, and highly stable radioimmunoconjugates. Furthermore, preclinical experiments in murine models of colorectal cancer have shown that these site-selectively labeled radioimmunoconjugates exhibit far superior in vivo performance compared to radiolabeled antibodies synthesized via maleimide-based conjugations. In this protocol, we will describe the four-step synthesis of PODS, the creation of a bifunctional PODS-bearing variant of the ubiquitous chelator DOTA (PODS-DOTA), and the conjugation of PODS-DOTA to the HER2-targeting antibody trastuzumab.

In this protocol, we will describe the synthesis of PODS, a phenyoxadiazolyl methyl sulfone-based reagent for the site-selective attachment of cargos to the thiols of biomolecules, particularly antibodies. In addition, we will describe the synthesis and characterization of a PODS-bearing bifunctional chelator and its conjugation to a model antibody.

硫醇反应试剂的合成与生物共轭作用--用于原位选择性修饰免疫共轭的生成

Maleimide-bearing bifunctional probes have been employed for decades for the site-selective modification of thiols in biomolecules, especially antibodies. ...

NMR-Based Activity Assays for Determining Compound Inhibition, IC50 Values, Artifactual Activity, and Whole-Cell Activity of Nucleoside Ribohydrolases

NMR spectroscopy is often used for the identification and characterization of enzyme inhibitors in drug discovery, particularly in the context of fragment screening. NMR-based activity assays are ideally suited to work at the higher concentrations of test compounds required to detect these weaker inhibitors. The dynamic range and chemical shift dispersion in an NMR experiment can easily resolve resonances from substrate, product, and test compounds. This contrasts with spectrophotometric assays, in which read-out interference problems often arise from compounds with overlapping UV-vis absorption profiles. In addition, since they lack reporter enzymes, the single-enzyme NMR assays are not prone to coupled-assay false positives. This attribute makes them useful as orthogonal assays, complementing traditional high throughput screening assays and benchtop triage assays. Detailed protocols are provided for initial compound assays at 500 &#956;M and 250 &#956;M, dose-response assays for determining IC50 values, detergent counter screen assays, jump-dilution counter screen assays, and assays in E. coli whole cells. The methods are demonstrated using two nucleoside ribohydrolase enzymes. The use of 1H NMR is shown for the purine-specific enzyme, while 19F NMR is shown for the pyrimidine-specific enzyme. The protocols are generally applicable to any enzyme where substrate and product resonances can be observed and distinguished by NMR spectroscopy. To be the most useful in the context of drug discovery, the final concentration of substrate should be no more than 2-3x its Km value. The choice of NMR experiment depends on the enzyme reaction and substrates available as well as available NMR instrumentation.

NMR-Based Activity Assays for Determining Compound Inhibition, IC50 Values, Artifactual Activity, and Whole-Cell Activity of Nucleoside Ribohydrolases

NMR-based activity assays have been developed to identify and characterize inhibitors of two nucleoside ribohydrolase enzymes. Protocols are provided for initial compound assays at 500 &#956;M and 250 &#956;M, dose-response assays for determining IC50 values, detergent counter screen assays, jump-dilution counter screen assays, and assays in E. coli whole cells.

基于NMR的活性测定,用于测定核苷裂解酶的化合物抑制、IC50值、伪影活性和全细胞活性

NMR spectroscopy is often used for the identification and characterization of enzyme inhibitors in drug discovery, particularly in the context of fragment ...