我们要感谢S.雷斯勒（博士，贝勒医学院分子与细胞生物学部）建议使用的蛋白质集中在细胞培养上清液中基质金属蛋白酶的浓度增加。 这个项目是由夫人祈福长老白格雷厄姆赋研究基金和美国国立卫生研究院/ NIAMS（AR059838）对CB的赠款支持。内容完全是作者的责任，并不一定代表美国国立卫生研究院的官方意见。

1。加载和运行的凝胶<ol><li>所有样品必须充分准备，保持酶的功能，采集后立即使用，或冷冻保存于-80 ° C。样品必须不包含还原剂（这样的β-巯基乙醇）或凝胶装载前煮沸。 </li><li>打开袋录像带里面含有一种凝胶，录像带用去离子水冲洗。 <ul><li>取出录像带的底部，并从凝胶顶部梳保护胶带。 </li><li>运行缓冲液（去离子水稀释至1X）冲洗井的三倍。 </li><li>凝胶放入微型电池，确保在录像带较小的侧面向内。凝胶张力楔锁定到位。迷你细胞允许并行运行一个或两个凝胶。 </li><li>填写与1X井水平以上运行缓冲区的顶部（内）室，检查有无渗漏。在泄漏的情况下，删除的缓冲区，并重新定位凝胶。 </li><li>下腔填充1X运行缓冲。 </li></ul></li><li>装入一个孔10μL蛋白质的分子标记。 <ul><li>等量混合成井使用凝胶加载提示（每个样品之​​间的改变）的凝胶，凝胶样缓冲液和样品和负载。这些井可装载至20μL总。 </li></ul></li><li>迷你细胞上的盖子和电极线连接到电源。开关电源，并设置它运行在125 V恒定为90分钟。检查下腔线的小气泡的形成，表明当前的流通。 </li><li>当运行您的第一个凝胶，迁移每15分钟监测方面取得的进展，使用溴酚蓝上样缓冲液作为一项指标包括。让凝胶运行，直到指示剂到达凝胶底部。 </li></ol> 2。复性和发展的凝胶<ol><li>对于每一个凝胶，准备1X复性缓冲和变性缓冲液200毫升，100毫升，无论是在去离子水。 </li><li>当溴酚蓝跟踪染料到达凝胶底部，接通电源关闭，打开迷你细胞，取出凝胶。双方在录像带使用的胶刀（或称重锅铲）分开。切一个角落，以纪念凝胶的方向。 </li><li>小心取出凝胶放入容器纸盒和地方与100毫升的复性缓冲。孵育30分钟，在室温下轻轻摇动。 </li><li>删除复性缓冲，加入100毫升凝胶发展缓冲区。孵育30分钟，在室温下轻轻摇动。 </li><li>删除发展中国家的缓冲区，并添加100毫升的发展缓冲的凝胶。孵育过夜（16-18小时），在37 ° C。 </li><li>删除发展中国家缓冲区，并在室温下轻轻摇动去离子水冲洗三次（每次5分钟）。 </li><li>扫描保存的蛋白质标准频段的精确定位，因为他们将成为少用或不可见的凝胶染色后的凝胶。 </li><li>染色，加入20毫升SimplyBlue Safestain的凝胶，凝胶。轻轻摇动的温度在室温下孵育1小时。 </li><li>卸下SimplyBlue SafeStain和色斑的100毫升或更多的去离子水凝胶在室温下轻轻摇动下一个小时。 </li><li>为了获得更好的效果，更换新鲜去离子水，并培育，再在室温下轻轻摇动下的小时或更长时间。 </li></ol> 3。数据分析<ol><li>小心取出凝胶，水和一张塑料布保护。 </li><li>扫描分辨率300 dpi或更高的凝胶。保存在TIFF格式的图像（图1A）。 </li><li> ImageJ（或其他类似的软件）测量的波段强度。 <ul><li>打开ImageJ（图1A）TIFF文件。 </li><li>在黑色和白色的直观选择“图像&gt;类型&gt; 8位”（图1B）。 </li><li>使用矩形选择工具，勾勒出的第一个乐队，至少两次高于宽（这是一个软件的要求）绘制一个矩形。 </li><li>按“1”或选择“分析&gt;凝胶&gt;选择”第一线“乐队将概述。 </li><li>将出现一个新的矩形，将其移动到下一个波段，选择“分析&gt;凝胶&gt;选择下一个车道”。重复，直到所有频段选择（图1B）。 </li><li>按“3”或选择“分析&gt;凝胶&gt;剧情通道”，生成的每个波段的个人资料（图1C）情节。 </li><li>使用直线选择（应该已经自动选择）绘制基准线，使利益的高峰，是一个完全封闭的区域。 </li><li>选择棒工具，并在每个高峰，单击以选中它。 </li><li>选择“分析&gt;凝胶&gt;标签峰”，以获得与每个选定的高峰领域的一个表。这些数据可以绘制成这样（Figure 1D）或可归到一个频带的价值。这正常化池几个复制凝胶的值，以确定结果的统计学意义时，显得尤为重要。 </li></ul></li></ol> 4。代表性的成果我们展示一个与不同的细胞培养上清中含有不同量的MMP - 2（图1A）的装入11井的凝胶。这种凝胶的直接观察表明在一些井之间的MMP - 2浓度明显的差异。例如，很显然，井＃4和5包含得多好＃11，甚至10号MMP - 2。客观量化的乐队，我们​​已经用ImageJ软件（图1B - D）密度测量，确认以及＃11和水井＃4和第5和一个约2倍的差异，在差异中MMP - 2的金额约4倍MMP - 2以及10号和水井＃4和5之间的数额。 <img src="/files/ftp_upload/2445/2445fig1.jpg" alt="图1" /> 图1。明胶酶谱法检测MMP - 2。一，扫描图像明胶凝胶。井数字表明在凝胶的顶部。 B，同在一个凝胶显示在黑色和白色为光密度。每个频带被选定在ImageJ的矩形。 A和B，光密度剖面的两个例子所示凝胶（带＃4和＃11）。直线绘制在每个峰的基地，创建一个封闭的区域。研发，绘制峰面积为A和B前（左）后（右）正常化带＃1的密度所示的凝胶。 <img src="/files/ftp_upload/2445/2445fig2.jpg" alt="图2" /> 图2这个数字演示如何酶谱可以作为一种半定量技术。我们已经加载在连续7口井连续稀释（按1:1稀释的步骤）和两个亲的MMP - 2和MMP - 2的带密度测量。

<ol>
	<li>Luo, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+matrix+metalloproteinases+in+the+morphogenesis+of+the+cerebellar+cortex.">The role of matrix metalloproteinases in the morphogenesis of the cerebellar cortex.</a> Cerebellum. 4, 239-245 (2005).</li><li>Pasternak, B., Aspenberg, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metalloproteinases+and+their+inhibitors+-+diagnostic+and+therapeutic+opportunities+in+orthopedics.">Metalloproteinases and their inhibitors - diagnostic and therapeutic opportunities in orthopedics.</a> Acta Orthop. 80, 693-703 (2009).</li><li>Kessenbrock, K., Plaks, V., Werb, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrix+metalloproteinases:+regulators+of+the+tumor+microenvironment.">Matrix metalloproteinases: regulators of the tumor microenvironment.</a> Cell. 141, 52-67 (2010).</li><li>Lagente, V., Boichot, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+matrix+metallopreoteinases+in+the+inflammatory+process+of+respiratory+diseases.">Role of matrix metallopreoteinases in the inflammatory process of respiratory diseases.</a> J. Mol. Cell. Cardiol. 48, 440-444 (2010).</li><li>Rodr&#237;guez, D., Morrison, C. J., Overall, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrix+metallopreoteinases:+what+do+they+not+do?+New+substrates+and+biological+roles+identified+by+murine+models+and+proteomics.">Matrix metallopreoteinases: what do they not do? New substrates and biological roles identified by murine models and proteomics.</a> Biochim. Biophys. Acta. 1803, 39-54 (2010).</li><li>Bourboulia, D., Stetler-Stevenson, W. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrix+metalloproteinases+(MMPs)+and+tissue+inhibitors+of+metalloproteinases+(TIMPs):+positive+and+negative+regulators+in+tumor+cell+adhesion.">Matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs): positive and negative regulators in tumor cell adhesion.</a> Semin. Cancer Biol. , (2010).</li><li>Heussen, C., Dowdle, E. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophoretic+analysis+of+plasminogen+activators+in+polyacrylamide+gels+containing+sodium+dodecyl+sulfate+and+copolymerized+substrates.">Electrophoretic analysis of plasminogen activators in polyacrylamide gels containing sodium dodecyl sulfate and copolymerized substrates.</a> Analytical Biochem. , 102-196 (1980).</li><li>Lombard, C., Saulnier, J., Wallach, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assays+of+matrix+metalloproteinases+(MMPs)+activities:+a+review.">Assays of matrix metalloproteinases (MMPs) activities: a review.</a> Biochimie. 87, 265-272 (2005).</li><li>Snoek-van Beurden, P. A., Von den Hoff, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zymographic+techniques+for+the+analysis+of+matrix+metalloproteinases+and+their+inhibitors.">Zymographic techniques for the analysis of matrix metalloproteinases and their inhibitors.</a> Biotechniques. 38, 73-83 (2005).</li><li>Kupai, K., Szucs, G., Cseh, S., Hajdu, I., Csonka, C., Csont, T., Ferdinandy, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrix+metalloproteinase+activity+assays:+importance+of+zymography.">Matrix metalloproteinase activity assays: importance of zymography.</a> J. Pharmacol. Toxicol. Methods. 61, 205-209 (2010).</li><li>Kleiner, D. E., Stetler-Stevenson, W. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+zymography:+detection+of+pictogram+quantities+of+gelatinases.">Quantitative zymography: detection of pictogram quantities of gelatinases.</a> Anal. Biochem. 218, 325-329 (1994).</li><li>Yu, W. H., Woessner, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heparin-enhanced+zymographic+detection+of+matrilysin+and+collagenases.">Heparin-enhanced zymographic detection of matrilysin and collagenases.</a> Anal. Biochem. 293, 38-42 (2001).</li></ol>

我们已经演示了如何执行zymographic分析细胞培养上清中MMP - 2的。 加载凝胶上的样品量必须根据凭经验确定的起源和MMP利益。负荷是否过小会防止检测，同时装载过多可能会导致饱和，因为只有这么多基板一种蛋白酶可以消化在乐队的领域。如果有必要，样品可以在去离子水稀释前与样缓冲液，或开发时间混合，可以减少从隔夜到4个小时。它也有可能集中在一个解决方案使用集中器（如Millipore公司目录＃UFC803024）的蛋白质。如果乐队仍隐约可见，它可能是必要制定一个较长时间的凝胶，甚至长达48小时。准备从组织培养上清样品时，应当指出，胎牛血清（和其他血清）中包含的蛋白酶可以影响的结果。出于这个原因，我们收集无血清培养基培养后，我们所有的上清。 在第2.9和2.10的协议中所描述的清洗需要删除的背景染色消化乐队。洗更长的时间将删除更多的背景，但也将暗淡的基材整个凝胶染色。如果需要更长的洗涤时间是必要的，我们建议扫描凝胶每隔几个小时，选择最好的对比度扫描。 这种技术可用于检测其他的蛋白酶。例如，可以检测到MMP - 9明胶凝胶体，也程度较低的MMP - 1，MMP - 8和MMP - 13，明胶是不是他们的首选基材。 MMP - 1和MMP - 13是最好的胶原蛋白的酶谱检测，而酪蛋白是首选基板和MMP - 11也可以检测MMP - 1，MMP - 3，MMP - 7，MMP - 12和MMP - 13 9。 MMP - 7（基质溶解）和胶原酶（MMP - 1和MMP - 13）是很难检测时，在低的水平目前在酪蛋白或明胶凝胶。此外肝素样品在凝胶加载时间显着提高MMP - 7的检测限。 MMP - 1和MMP - 13，肝素需要被添加到凝胶电泳运行后已经正在进行12。 由于酶谱措施酶的活性酶的变性和复性后，它会测量所有样品中的基质金属蛋白酶的活动。这包括酶，亲酶，并绑定到内源性抑制剂的酶（如MMP - 2）。不过，可以通过比较带密度的活性酶和酶，这将有一个稍高的分子量来确定样品中的基质金属蛋白酶激活水平。 酶谱法往往是不足够的识别MMP。 MMP与已知分子量标准的迁移水平的比较，帮助识别，但应该指出，这些标准包含还原剂和非还原条件下使用时，他们可能会表示不同分子量9。一个选项是一种可溶性的重组MMP的负载测试样品相同凝胶。然而，与其他蛋白质基质金属蛋白酶可能诱导表观分子量的变化。选择性MMP抑制剂可以被添加到发展缓冲区作为药理工具孵化期间的凝胶（或部分削减了一半的凝胶）的身份interest.A第二个方法（免疫印迹，免疫细胞化学法或ELISA）MMP建议帮助鉴定MMP利益。应该指出，Western印迹检测限制通常比zymographic凝胶低得多，可能会导致假阴性结果使用这种技术时。

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Xcell SureLock Mini-Cell CE mark electrophoresis apparatus</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>EI0001</td>
<td>&nbsp;</td>
<tr>
<td>Power supply (model 302)</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>93000-744</td>
<td>&nbsp;</td>
<tr>
<td>Novex 10% gelatin zymogram gels, 1.0 mm, 12 wells</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>EC61752BOX</td>
<td>&nbsp;</td>
<tr>
<td>Blue Juice gel loading buffer</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>10816015</td>
<td>&nbsp;</td>
<tr>
<td>Gel loading tips</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>53509-015</td>
<td>&nbsp;</td>
<tr>
<td>Protein molecular weight standard</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>LC5800</td>
<td>&nbsp;</td>
<tr>
<td>Novex Tris-Glycine-SDS running buffer</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>LC2675</td>
<td>&nbsp;</td>
<tr>
<td>Novex zymogram renaturing buffer</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>LC2670</td>
<td>&nbsp;</td>
<tr>
<td>Novex zymogram developing buffer</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>LC2671</td>
<td>&nbsp;</td>
<tr>
<td>SimplyBlue SafeStain</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>LC6060</td>
<td>&nbsp;</td>
<tr>
<td>Epson Perfection 4490 Photo Scanner</td>
<td>&nbsp;</td>
<td>Amazon</td>
<td>n/a</td>
<td>&nbsp;</td>
<tr>
<td>ImageJ software</td>
<td>&nbsp;</td>
<td>http://rsbweb.nih.gov/ij/ </td>
<td>n/a</td>
<td>Authored by W. Rasband, NIH/NIMH</td>
</tr>		</tbody>
		</table>
		</div>

detection of functional matrix metalloproteinases by zymography

基质金属蛋白酶（MMPs）是含有锌endopeptidases。他们降解蛋白质肽键的断裂。超过20基质金属蛋白酶已经确定，并分隔成六个小组，根据它们的结构和底物特异性（胶原酶，明胶酶，膜型[MT - MMP，stromelysins，matrilysins，和其他人）。基质金属蛋白酶在细胞的浸润，软骨退化，组织重构，伤口愈合和胚胎发育中发挥关键作用。因此，他们在正常的进程，并在许多疾病，如类风湿关节炎，癌症，慢性阻塞性肺疾病 1-6发病的参与。在这里，我们将重点对MMP - 2（明胶酶A，IV型胶原酶），基质金属蛋白酶广泛表达。我们将演示如何检测细胞培养上清液中MMP - 2的酶谱法，一种常用的，简单，但非常敏感的技术，在1980年首次描述C. Heussen 和 EB Dowdle 7-10。这种技术是半定量的，因此它可以被用于确定测试样品基质金属蛋白酶的重组MMP的已知浓度的水平时，装载在同一凝胶11。 聚丙烯酰胺凝胶含有十二烷基硫酸钠（SDS，线性的蛋白质）和明胶（MMP - 2的基板）装上含有基质金属蛋白酶（如细胞培养上清液，尿液或血清）的解决方案。样本缓冲区的目的是为了增加样品粘度（以方便装载凝胶），提供追踪染料（溴酚蓝;监测样本迁移），提供变性分子（线性蛋白质），并控制样品的pH值。蛋白质，然后允许电流下的迁移，在设计，以提供一个恒定的迁移率一个运行缓冲。迁移距离呈负相关的蛋白质的分子量（小分子蛋白通过凝胶中移动速度更快的比大的蛋白质，因此进一步下降凝胶迁移）。迁移后，凝胶放置在复性缓冲液使蛋白质，以重新获得接受高等教育的结构，酶的活性所必需的。凝胶，然后放置在一个发展中的缓冲区设计，允许蛋白酶消化其底。发展中的缓冲区也包含P - aminophenylmercuric醋酸（APMA）变为主动的基质金属蛋白酶激活非蛋白水解的基质金属蛋白酶。下一步，包括染色基板（在我们的例子明胶）。洗去多余的染料凝胶后，蛋白酶消化领域出现清晰的条带。乐队的更清晰，更集中的蛋白酶它包含。带染色强度可以通过光密度确定，如ImageJ软件使用，允许样品比较。

Watch this Scientific Journal Video about Detection of Functional Matrix Metalloproteinases by Zymography at JoVE.com

Detection of Functional Matrix Metalloproteinases by Zymography

本协议描述了一个用于检测培养上清或体液基质金属蛋白酶的活动为基础的检测。

酶谱法检测功能的基质金属蛋白酶

Matrix metalloproteinases (MMPs) are zinc-containing endopeptidases. They degrade proteins by cleavage of peptide bonds. More than twenty MMPs have been ...

detection-of-functional-matrix-metalloproteinases-by-zymography

Research

JoVE Journal

Biology

83.5K 次观看.  Baylor College of Medicine. 本协议描述了一个用于检测培养上清或体液基质金属蛋白酶的活动为基础的检测。

视频: Detection of Functional Matrix Metalloproteinases by Zymography

Labeling Stem Cells with Fluorescent Dyes for non-invasive Detection with Optical Imaging

Optical imaging (OI) is an easy, fast and inexpensive tool for in vivo monitoring of new stem cell based therapies. The technique is based on ex vivo labeling of stem cells with a fluorescent dye, subsequent intravenous injection of the labeled cells and visualization of their accumulation in specific target organs or pathologies. The presented technique demonstrates how we label human mesenchymal stem cells (hMSC) by simple incubation with the lipophilic fluorescent dye DiD (C67H103CIN2O3S) and how we label human embryonic stem cells (hESC) with the FDA approved fluorescent dye Indocyanine Green (ICG). The uptake mechanism is via adherence and diffusion of the lypophilic dye across the phospholipid cell membrane bilayer. The labeling efficiency is usually improved if the cells are incubated with the dye in serum-free media as opposed to incubation in serum-containing media. Furthermore, the addition of the transfection agent Protamine Sulfate significantly improves contrast agent uptake.

This video shows techniques for labeling of human embryonic stem cells and mesenchymal stem cells with fluorescent dyes. This technique can be used for an in vivo tracking of transplanted stem cells with optical imaging and for histopathological correlations with fluorescence microscopy.

与非侵入性检测荧光染料标记的干细胞与光学成像

Optical imaging (OI) is an easy, fast and inexpensive tool for in vivo monitoring of new stem cell based therapies. The technique is based on ex vivo ...

科研

生物学

Detection of Post-translational Modifications on Native Intact Nucleosomes by ELISA

The genome of eukaryotes exists as chromatin which contains both DNA and proteins. The fundamental unit of chromatin is the nucleosome, which contains 146 base pairs of DNA associated with two each of histones H2A, H2B, H3, and H41. The N-terminal tails of histones are rich in lysine and arginine and are modified post-transcriptionally by acetylation, methylation, and other post-translational modifications (PTMs). The PTM configuration of nucleosomes can affect the transcriptional activity of associated DNA, thus providing a mode of gene regulation that is epigenetic in nature 2,3. We developed a method called nucleosome ELISA (NU-ELISA) to quantitatively determine global PTM signatures of nucleosomes extracted from cells. NU-ELISA is more sensitive and quantitative than western blotting, and is useful to interrogate the epiproteomic state of specific cell types. This video journal article shows detailed procedures to perform NU-ELISA analysis.

Nucleosome ELISA (NU-ELISA) is a sensitive and quantitative method to detect global patterns of post-translational modifications in preparations of native, intact nucleosomes. These modifications include methylations, acetylations, and phosphorylations at specific histone amino acid residues, and hence NU-ELISA provides a global proteomic assay of the overall chromatin modification states of specific cell types.

检测采用ELISA法的母语完整的核小体的翻译后修饰

The genome of eukaryotes exists as chromatin which contains both DNA and proteins. The fundamental unit of chromatin is the nucleosome, which contains 146 ...

Harvesting and Cryo-cooling Crystals of Membrane Proteins Grown in Lipidic Mesophases for Structure Determination by Macromolecular Crystallography

An important route to understanding how proteins function at a mechanistic level is to have the structure of the target protein available, ideally at atomic resolution. Presently, there is only one way to capture such information as applied to integral membrane proteins (Figure 1), and the complexes they form, and that method is macromolecular X-ray crystallography (MX). To do MX diffraction quality crystals are needed which, in the case of membrane proteins, do not form readily. A method for crystallizing membrane proteins that involves the use of lipidic mesophases, specifically the cubic and sponge phases1-5, has gained considerable attention of late due to the successes it has had in the G protein-coupled receptor field6-21 (<a href="http://www.mpdb.tcd.ie" target="_blank">www.mpdb.tcd.ie</a>). However, the method, henceforth referred to as the in meso or lipidic cubic phase method, comes with its own technical challenges. These arise, in part, due to the generally viscous and sticky nature of the lipidic mesophase in which the crystals, which are often micro-crystals, grow. Manipulating crystals becomes difficult as a result and particularly so during harvesting22,23. Problems arise too at the step that precedes harvesting which requires that the glass sandwich plates in which the crystals grow (Figure 2)24,25 are opened to expose the mesophase bolus, and the crystals therein, for harvesting, cryo-cooling and eventual X-ray diffraction data collection.
The cubic and sponge mesophase variants (Figure 3) from which crystals must be harvested have profoundly different rheologies4,26. The cubic phase is viscous and sticky akin to a thick toothpaste. By contrast, the sponge phase is more fluid with a distinct tendency to flow. Accordingly, different approaches for opening crystallization wells containing crystals growing in the cubic and the sponge phase are called for as indeed different methods are required for harvesting crystals from the two mesophase types. Protocols for doing just that have been refined and implemented in the Membrane Structural and Functional Biology (MS&amp;FB) Group, and are described in detail in this JoVE article (Figure 4). Examples are given of situations where crystals are successfully harvested and cryo-cooled. We also provide examples of cases where problems arise that lead to the irretrievable loss of crystals and describe how these problems can be avoided. In this article the Viewer is provided with step-by-step instructions for opening glass sandwich crystallization wells, for harvesting and for cryo-cooling crystals of membrane proteins growing in cubic and in sponge phases.

Herein is described procedures implemented in the Caffrey Membrane Structural and Functional Biology Group to harvest and cryo-cool membrane protein crystals grown in lipidic cubic and sponge phases for use in structure determination using macromolecular X-ray crystallography.

在油脂中间相结构的测定生物大分子晶体生长的膜蛋白晶体收获和低温冷

An important route to understanding how proteins function at a mechanistic level is to have the structure of the target protein available, ideally at ...

Detection of Viral RNA by Fluorescence in situ Hybridization (FISH)

Viruses that infect cells elicit specific changes to normal cell functions which serve to divert energy and resources for viral replication. Many aspects of host cell function are commandeered by viruses, usually by the expression of viral gene products that recruit host cell proteins and machineries. Moreover, viruses engineer specific membrane organelles or tag on to mobile vesicles and motor proteins to target regions of the cell (during de novo infection, viruses co-opt molecular motor proteins to target the nucleus; later, during virus assembly, they will hijack cellular machineries that will help in the assembly of viruses). Less is understood on how viruses, in particular those with RNA genomes, coordinate the intracellular trafficking of both protein and RNA components and how they achieve assembly of infectious particles at specific loci in the cell. The study of RNA localization began in earlier work. Developing lower eukaryotic embryos and neuronal cells provided important biological information, and also underscored the importance of RNA localization in the programming of gene expression cascades. The study in other organisms and cell systems has yielded similar important information. Viruses are obligate parasites and must utilise their host cells to replicate. Thus, it is critical to understand how RNA viruses direct their RNA genomes from the nucleus, through the nuclear pore, through the cytoplasm and on to one of its final destinations, into progeny virus particles 1.
FISH serves as a useful tool to identify changes in steady-state localization of viral RNA. When combined with immunofluorescence (IF) analysis 22, FISH/IF co-analyses will provide information on the co-localization of proteins with the viral RNA3. This analysis therefore provides a good starting point to test for RNA-protein interactions by other biochemical or biophysical tests 4,5, since co-localization by itself is not enough evidence to be certain of an interaction. In studying viral RNA localization using a method like this, abundant information has been gained on both viral and cellular RNA trafficking events 6. For instance, HIV-1 produces RNA in the nucleus of infected cells but the RNA is only translated in the cytoplasm. When one key viral protein is missing (Rev) 7, FISH of the viral RNA has revealed that the block to viral replication is due to the retention of the HIV-1 genomic RNA in the nucleus 8. 
Here, we present the method for visual analysis of viral genomic RNA in situ. The method makes use of a labelled RNA probe. This probe is designed to be complementary to the viral genomic RNA. During the in vitro synthesis of the antisense RNA probe, the ribonucleotide that is modified with digoxigenin (DIG) is included in an in vitro transcription reaction. Once the probe has hybridized to the target mRNA in cells, subsequent antibody labelling steps (Figure 1) will reveal the localization of the mRNA as well as proteins of interest when performing FISH/IF.

Detection of Viral RNA by Fluorescence in situ Hybridization FISH

A fluorescence in situ hybridization (FISH) method was developed to visually detect viral genomic RNA using fluorescence microscopy. A probe is made with specificity to the viral RNA that can then be identified using a combination of hybridization and immunofluorescence techniques. This technique offers the advantage of identifying the localization of the viral RNA or DNA at steady-state, providing information on the control of intracellular virus trafficking events.

荧光检测病毒RNA<em&gt;原位</em&gt;杂交（FISH）

Viruses  that infect cells elicit specific changes to normal cell functions which serve  to divert energy and resources for viral replication. Many ...

Improving the Success Rate of Protein Crystallization by Random Microseed Matrix Screening

Random microseed matrix screening (rMMS) is a protein crystallization technique in which seed crystals are added to random screens. By increasing the likelihood that crystals will grow in the metastable zone of a protein's phase diagram, extra crystallization leads are often obtained, the quality of crystals produced may be increased, and a good supply of crystals for data collection and soaking experiments is provided. Here we describe a general method for rMMS that may be applied to either sitting drop or hanging drop vapor diffusion experiments, established either by hand or using liquid handling robotics, in 96-well or 24-well tray format.

Here we describe a general method for random microseed matrix screening. This technique is shown to significantly increase the success rate of protein crystallization screening experiments, reduce the need for optimization, and provide a reliable supply of crystals for data collection and ligand-soaking experiments.

提高蛋白质结晶的成功率随机Microseed矩阵筛选

Random microseed matrix screening  (rMMS) is a protein crystallization technique in which seed crystals are added  to random screens. By increasing the ...

Detection of Live Escherichia coli O157:H7 Cells by PMA-qPCR

A unique open reading frame (ORF) Z3276 was identified as a specific genetic marker for E. coli O157:H7. A qPCR assay was developed for detection of E. coli O157:H7 by targeting ORF Z3276. With this assay, we can detect as low as a few copies of the genome of DNA of E. coli O157:H7. The sensitivity and specificity of the assay were confirmed by intensive validation tests with a large number of E. coli O157:H7 strains (n = 369) and non-O157 strains (n = 112). Furthermore, we have combined propidium monoazide (PMA) procedure with the newly developed qPCR protocol for selective detection of live cells from dead cells. Amplification of DNA from PMA-treated dead cells was almost completely inhibited in contrast to virtually unaffected amplification of DNA from PMA-treated live cells. Additionally, the protocol has been modified and adapted to a 96-well plate format for an easy and consistent handling of a large number of samples. This method is expected to have an impact on accurate microbiological and epidemiological monitoring of food safety and environmental source.

Detection of Live Escherichia coli O157:H7 Cells by PMA-qPCR

A qPCR assay was developed for detection of Escherichia coli O157:H7 targeting a unique genetic marker, Z3276. The qPCR was combined with propidium monoazide (PMA) treatment for live cell detection. This protocol has been modified and adapted to a 96-well plate format for easy and consistent handling of numerous samples

检测现场的<em&gt;大肠杆菌</em&gt; O157：H7细胞的PMA-qPCR的

A unique open reading frame (ORF) Z3276 was identified as a specific genetic marker for E. coli O157:H7. A qPCR assay was developed for detection of E. ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

在造血干细胞和祖细胞的成年小鼠颅骨骨髓体内四维跟踪

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Fecal Glucocorticoid Analysis: Non-invasive Adrenal Monitoring in Equids

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, corticotrophin releasing hormone (CRH) is released from the hypothalamus in the brain. This stimulates the release of adrenocorticotrophic hormone (ACTH) from the pituitary gland, which in turn stimulates release of glucocorticoids from the adrenal gland. In horses the glucocorticoid corticosterone is responsible for several adaptations needed to support equine flight behaviour and subsequent removal from the aversive situation. Corticosterone metabolites can be detected in the feces of horses and assessment offers a non-invasive option to evaluate long term patterns of adrenal activity. Fecal assessment offers advantages over other techniques that monitor adrenal activity including blood plasma and saliva analysis. The non-invasive nature of the method avoids sampling stress which can confound results. It also allows the opportunity for repeated sampling over time and is ideal for studies in free ranging horses. This protocol describes the enzyme linked immunoassay (EIA) used to assess feces for corticosterone, in addition to the associated biochemical validation.

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. The method offers a non-invasive option to assess long term patterns in both domestic and free ranging horses. This protocol describes the enzyme linked immunoassay involved and the associated biochemical validation.

糖皮质激素粪分析：在马属动物无创监测肾上腺

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, ...

Detection of RNA-binding Proteins by In Vitro RNA Pull-down in Adipocyte Culture

RNA-binding proteins (RBPs) are emerging as a regulatory layer in the development and function of adipose. RBPs play a key role in the gene expression regulation at posttranscriptional levels by affecting the stability and translational efficiency of target mRNAs. RNA pull-down technique has been widely used to study RNA-protein interaction, which is necessary to elucidate the mechanism underlying RBPs&#39; as well as long non-coding RNAs&#39; (lncRNAs) function. However, the high lipid abundance in adipocytes poses a technical challenge in conducting this experiment. Here a detailed RNA pull-down protocol is optimized for primary adipocyte culture. An RNA fragment from androgen receptor&#39;s (AR) 3&#39; untranslated region (3&#39;UTR) containing an adenylate-uridylate-rich elementwas used as an example to demonstrate how to retrieve its RBP partner, HuR protein, from adipocyte lystate. The method described here can be applied to detect the interactions between RBPs and noncoding RNAs, as well as between RBPs and coding RNAs.

Detection of RNA-binding Proteins by In Vitro RNA Pull-down in Adipocyte Culture

An RNA pull-down protocol is optimized here for detection of interactions between RNA-binding proteins (RBPs) and noncoding as well as coding RNAs. An RNA fragment from androgen receptor (AR) was used as an example to demonstrate how to retrieve its RBP from lystate of primary brown adipocytes.

RNA结合蛋白的检测通过<em&gt;体外</em在脂肪细胞培养&gt; RNA下拉

RNA-binding proteins (RBPs) are emerging as a regulatory layer in the development and function of adipose. RBPs play a key role in the gene expression ...

A Rapid, Scalable Method for the Isolation, Functional Study, and Analysis of Cell-derived Extracellular Matrix

The extracellular matrix (ECM) is recognized as a diverse, dynamic, and complex environment that is involved in multiple cell-physiological and pathological processes. However, the isolation of ECM, from tissues or cell culture, is complicated by the insoluble and cross-linked nature of the assembled ECM and by the potential contamination of ECM extracts with cell surface and intracellular proteins. Here, we describe a method for use with cultured cells that is rapid and reliably removes cells to isolate a cell-derived ECM for downstream experimentation.
Through use of this method, the isolated ECM and its components can be visualized by in situ immunofluorescence microscopy. The dynamics of specific ECM proteins can be tracked by tracing the deposition of a tagged protein using fluorescence microscopy, both before and after the removal of cells. Alternatively, the isolated ECM can be extracted for biochemical analysis, such as sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. At larger scales, a full proteomics analysis of the isolated ECM by mass spectrometry can be conducted. By conducting ECM isolation under sterile conditions, sterile ECM layers can be obtained for functional or phenotypic studies with any cell of interest. The method can be applied to any adherent cell type, is relatively easy to perform, and can be linked to a wide repertoire of experimental designs.

The extracellular matrix plays a major role in defining the microenvironment of cells and in modulating cell behavior and phenotype. We describe a rapid method for the isolation of cell-derived extracellular matrix, which can be adapted to different scales for microscopic, biochemical, proteomic, or functional studies.