我们感谢有益的意见A.列奇和念稿子。这项工作是支持的医学研究理事会（MRC）的拨款，细胞生物学组和一个人类前沿科学计划资助（RGP0048/2009-C）。

1。酶促反应 - 第1天（1小时在下午） <ol><li>第一步是准备10-20独立的酶反应， 其中的IP 6 K1或VIP1 的转换IP 6 pyrophosphorylated亚型。 </li><li>我们使用IP的6 K1和GST - VIP1酶纯化，从E.根据该协议的大肠杆菌以前描述 17,18 。 </li><li>准备50μL的反应，含有1X反应缓冲液（30毫米HEPES pH为6.8，50毫米氯化钠，硫酸镁6毫米，1毫米的数码地面电视），6毫米磷酸（PCR），25 U / mL的CreatinePhosphoKinase（CPK），5毫米（ ATP MG盐），IP6的0.3毫米，0.05〜0.1微克他的IP 6 K1或GST - VIP1。调整音量与MiliQ DDH 2 O </li><li>简要地旋转在37 ° C过夜旋转的反应和孵化。 </li></ol> 2。聚丙烯酰胺凝胶铸造和加载 - 第2天（4个小时，下午） <ol><li>聚丙烯酰胺凝胶是准备用24厘米长，18厘米宽的玻璃板和1.5毫米宽的间隔。通常使用16线或单线制备梳。 </li><li>准备一个组合（50毫升/凝胶）包含以下内容:35.5％（W / V）丙烯酰胺:双丙烯酰胺19:1，1X三/硼酸/ EDTA（TBE），0.05％（W / V）过硫酸铵（APS ），0.05％（W / V）TEMED。倒入混合前播玻璃板之间，插入梳子，让聚合为30-60分钟，在室温下。 </li><li>一旦凝胶聚合，转让设备冷藏室和1X TBE约30-60分钟200-300伏前运行。 </li><li>添加OrangeG染料1X（10毫米的Tris -盐酸pH7.0的，1 mM的EDTA，30％甘油，0.1％OrangeG）到每一个反应。准备样品含有2的IP 6 nmol负载作为一个标准的控制。 </li><li>运行缓冲液使用的注射器和一个21G的针头，以消除任何沉淀的每口井彻底洗净，然后装入凝胶。避免加载上侧井。 </li><li>运行在450-550伏特（7 MAMP /凝胶）过夜的凝胶，凝胶底部在过去的10厘米，直到OrangeG染料带。 </li></ol> 3。隔离的IP 7 -第3天（4小时）和第4天（6-7小时SpeedVac干燥过程） <ol><li>拆开凝胶仪器，小心地取出一个玻璃板上留下另一种凝胶。剪切从正上方OrangeG染料带的一小部分的凝胶底部包含的IP标准和一个样本巷， 如图 1所示。 </li><li>溅污几分钟凝胶甲苯胺蓝（0.1％（W / V），20％（W / V）甲醇，甲苯胺蓝2％（W ​​/ V）甘油）的削减部分（1-3分钟）或直到肌醇焦带出现。以前删除以防止干燥的未染色的凝胶，凝胶的顶部，把玻璃板。 </li></ol> IP 7段应该是可见的，因为它运行速度略慢 6比IP标准。三磷酸腺苷，运行速度比IP 6，也应可见（ 图1）。传输的凝胶染色部分中去染色溶液（20％（W / V）的甲醇）为几分钟，洗去多余的甲苯胺蓝和重新定位与未染色的凝胶，凝胶。 如果需要更高pyrophosphorylated肌醇亚型（IP和IP 9）可视化，与甲苯胺蓝染色溶液染色凝胶在室温为20分钟。随后，洗去了约15分钟的染色液甲苯胺蓝。 <ol start="3"><li>用刀片切割未染色的部分作为参考，使用凝胶染色凝胶（ 图1）确定的IP 7迁移位置上的IP 7段。 </li><li>把IP 的7段，从凝胶削减15毫升管，并添加10毫升的MilliQ DDH 2 O为10分钟，在室温下放置在旋转管。丢弃删除超过TBE和微观的丙烯酰胺颗粒的液体。 </li><li>随后，执行两个脱水水化周期。加入5毫升50％（W / V）甲醇管与包含IP 7凝胶，并在室温下2小时旋转。转移到一个新的15 mL的试管中，MilliQ DDH 2 O 5毫升的凝胶片，并在室温下2小时旋转。不要丢弃的甲醇和管MilliQ H 2 O。重新转移在以前使用的15 mL管的聚丙烯酰胺凝胶的脱水水化周期重复一次。洗，可在夜间进行。 </li><li>要集中洗脱IP 7，一起干，10毫升（5毫升MilliQddH 2 O和5毫升50％（W / V）甲醇）在60 ° C的加热一个SpeedVac </li><li>一旦样品近干，转移剩余的液体（300〜600μL）± 1.5 mL离心管中，2分钟5000转的旋转。 </li><li>收集上清转移到一个新的1.5 mL离心管;离开底部20-30μL，因为它可能含有丙烯酰胺颗粒。 </li><li>如果必要的话，继续使用不加热Speed​​Vac干燥过程。复苏的IP 7大大减少，如果样品完全干燥，因此终止干燥过程中，当样品达到100-300μL的体积。 </li></ol> 4。 IP 7浓度和纯度的测定。 <ol><li>使用2-5μL恢复的IP 7示例运行页面上的凝胶，同样第2.2-2.5。载入几个稀释浓度的标准和4 nmol聚磷标记的IP 6（即0.5，1，2，4 nmol）。运行后的凝胶，可视化染色和甲苯胺蓝溶液染色整个凝胶肌醇焦亚型，在3.2节（ 图2A）条所述的程序。 </li><li>苯胺染色后，浓度可通过扫描凝胶，比较之间的IP 6 7和IP强度差异，利用图像处理软件，如图像- J，如图2b所示。 </li></ol> 5。代表性的成果: 制备的IP 6 7到IP使用IP 6 K1和VIP1的酶的酶转化，都可以轻松解决使用PAGE分析（ 图 1 ）。 IP作为规模控制的 6甲苯胺蓝凝胶染色装载允许pyrophosphorylated衍生物的识别，因为它们运行速度慢，肌醇环上的磷酸基团的数量而定。上述过程，允许方便的IP 7净化。分析纯化的肌醇焦逐页显示我们的IP 7（ 图2A）的纯度。有趣的是，IP 7 VIP1的产品1/3PP-IP 5异构体的迁移比5PP - IP的IP 7 5异构体，是由IP 6 K1产生慢。使用的IP 6标准允许浓度纯化的IP 7（ 图2B）容易量化。在进一步的实验中使用的 IP 7，其生物活性，可评估 （ 图3）。 5PP - IP 5孵育VIP1的IP 7磷酸DDP1（diphosphoinositol多聚磷酸酯phosphohydrolase）。常规纯化的IP地址转换为IP VIP1 8 DDP1 6到IP（ 图3） 。 <img alt="图1" src="/files/ftp_upload/3027/3027fig1.jpg" /> 图1:页面和隔离的IP 7波段苯胺染色包含标准（IP）的凝胶部分被削减和使用甲苯胺蓝溶液染色。三个频段（从上到下）6 IP 7，IP和ATP。然后对准剩余的凝胶的凝胶染色部分。这使得凝胶含有部分IP 7，这样就可以削减和纯化（虚线框）的本地化。 <img alt="图2" src="/files/ftp_upload/3027/3027fig2.jpg" /> 图2:IP 6 K1和VIP1的反应产物进行分析 。 a）分析甲苯胺蓝染色的IP 6（4，2，1，0.5 nmol）使用，以确定两个IP 6 K1（5PP - IP 5）和VIP1（1/3PP-IP纯化的IP 7浓度5）反应，B）散点图分析，以确定浓度纯化的IP 7。浓度进行了测定，根据带的强度，使用imageJ软件计算，比预先确定的金额的IP 6 。 X轴代表强度，Y轴代表在nmol表示的浓度。 <img alt="图3" src="/files/ftp_upload/3027/3027fig3.jpg" /> 图3:IP 7生物活性的分析，以确定纯化的IP 7的质量，我们培养与VIP1 或与IP 7磷酸DDP1 5PP - IP（IP 6 K1 生成的IP 7）页上的反应，然后解决。 DAPI染色和甲苯胺染色显示8的IP由VIP1预期的生产和转换的IP 7 DDP1的IP 6 。

<ol>
	<li>Bennett, M., Onnebo, S. M., Azevedo, C., Saiardi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inositol+pyrophosphates:+metabolism+and+signaling.">Inositol pyrophosphates: metabolism and signaling.</a> Cell Mol Life Sci. 63, 552-564 (2006).</li><li>Burton, A., Hu, X., Saiardi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Are+inositol+pyrophosphates+signalling+molecules?.">Are inositol pyrophosphates signalling molecules?.</a> J Cell Physiol. 220, 8-15 (2009).</li><li>Barker, C. J., Illies, C., Gaboardi, G. C., Berggren, P. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inositol+pyrophosphates:+structure,+enzymology+and+function.">Inositol pyrophosphates: structure, enzymology and function.</a> Cell Mol Life Sci. 66, 3851-3871 (2009).</li><li>Shears, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diphosphoinositol+polyphosphates:+metabolic+messengers.">Diphosphoinositol polyphosphates: metabolic messengers.</a> Mol Pharmacol. 76, 236-252 (2009).</li><li>Saiardi, A., Erdjument-Bromage, H., Snowman, A. M., Tempst, P., Snyder, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+of+diphosphoinositol+pentakisphosphate+by+a+newly+identified+family+of+higher+inositol+polyphosphate+kinases.">Synthesis of diphosphoinositol pentakisphosphate by a newly identified family of higher inositol polyphosphate kinases.</a> Curr Biol. 9, 1323-1326 (1999).</li><li>Draskovic, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inositol+hexakisphosphate+kinase+products+contain+diphosphate+and+triphosphate+groups.">Inositol hexakisphosphate kinase products contain diphosphate and triphosphate groups.</a> Chem Biol. 15, 274-286 (2008).</li><li>Mulugu, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+conserved+family+of+enzymes+that+phosphorylate+inositol+hexakisphosphate.">A conserved family of enzymes that phosphorylate inositol hexakisphosphate.</a> Science. 316, 106-109 (2007).</li><li>Fridy, P. C., Otto, J. C., Dollins, D. E., York, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cloning+and+characterization+of+two+human+VIP1-like+inositol+hexakisphosphate+and+diphosphoinositol+pentakisphosphate+kinases.">Cloning and characterization of two human VIP1-like inositol hexakisphosphate and diphosphoinositol pentakisphosphate kinases.</a> J Biol Chem. 282, 30754-30762 (2007).</li><li>Illies, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Requirement+of+inositol+pyrophosphates+for+full+exocytotic+capacity+in+pancreatic+beta+cells.">Requirement of inositol pyrophosphates for full exocytotic capacity in pancreatic beta cells.</a> Science. 318, 1299-1302 (2007).</li><li>Saiardi, A., Resnick, A. C., Snowman, A. M., Wendland, B., Snyder, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inositol+pyrophosphates+regulate+cell+death+and+telomere+length+through+phosphoinositide+3-kinase-related+protein+kinases.">Inositol pyrophosphates regulate cell death and telomere length through phosphoinositide 3-kinase-related protein kinases.</a> Proc Natl Acad Sci U S A. 102, 1911-1914 (2005).</li><li>York, S. J., Armbruster, B. N., Greenwell, P., Petes, T. D., York, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inositol+diphosphate+signaling+regulates+telomere+length.">Inositol diphosphate signaling regulates telomere length.</a> J Biol Chem. 280, 4264-4269 (2005).</li><li>Luo, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inositol+pyrophosphates+mediate+chemotaxis+in+Dictyostelium+via+pleckstrin+homology+domain-PtdIns(3,4,5)P3+interactions.">Inositol pyrophosphates mediate chemotaxis in Dictyostelium via pleckstrin homology domain-PtdIns(3,4,5)P3 interactions.</a> Cell. 114, 559-572 (2003).</li><li>Saiardi, A., Sciambi, C., McCaffery, J. M., Wendland, B., Snyder, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inositol+pyrophosphates+regulate+endocytic+trafficking.">Inositol pyrophosphates regulate endocytic trafficking.</a> Proc Natl Acad Sci U S A. 99, 14206-14211 (2002).</li><li>Auesukaree, C., Tochio, H., Shirakawa, M., Kaneko, Y., Harashima, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plc1p,+Arg82p,+and+Kcs1p,+enzymes+involved+in+inositol+pyrophosphate+synthesis,+are+essential+for+phosphate+regulation+and+polyphosphate+accumulation+in+Saccharomyces+cerevisiae.">Plc1p, Arg82p, and Kcs1p, enzymes involved in inositol pyrophosphate synthesis, are essential for phosphate regulation and polyphosphate accumulation in Saccharomyces cerevisiae.</a> J Biol Chem. 280, 25127-25133 (2005).</li><li>Azevedo, C., Burton, A., Ruiz-Mateos, E., Marsh, M., Saiardi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inositol+pyrophosphate+mediated+pyrophosphorylation+of+AP3B1+regulates+HIV-1+Gag+release.">Inositol pyrophosphate mediated pyrophosphorylation of AP3B1 regulates HIV-1 Gag release.</a> Proc Natl Acad Sci U S A. 106, 21161-21166 (2009).</li><li>Bhandari, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+pyrophosphorylation+by+inositol+pyrophosphates+is+a+posttranslational+event.">Protein pyrophosphorylation by inositol pyrophosphates is a posttranslational event.</a> Proc Natl Acad Sci U S A. 104, 15305-15310 (2007).</li><li>Azevedo, C., Burton, A., Bennett, M., Onnebo, S. M., Saiardi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+of+InsP7+by+the+Inositol+Hexakisphosphate+Kinase+1+(IP6K1).">Synthesis of InsP7 by the Inositol Hexakisphosphate Kinase 1 (IP6K1).</a> Methods Mol Biol. 645, 73-85 (2010).</li><li>Otto, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biochemical+analysis+of+inositol+phosphate+kinases.">Biochemical analysis of inositol phosphate kinases.</a> Methods Enzymol. 434, 171-185 (2007).</li><li>Losito, O., Szijgyarto, Z., Resnick, A. C., Saiardi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inositol+pyrophosphates+and+their+unique+metabolic+complexity:+analysis+by+gel+electrophoresis.">Inositol pyrophosphates and their unique metabolic complexity: analysis by gel electrophoresis.</a> PLoS One. 4, e5580-e5580 (2009).</li></ol>

没有利益冲突的声明。

在生物化学肌醇焦的使用受到严格限制商业得不到这种化合物与现有的检测方法的敏感性差。的页面，从而使拥有不同数量的磷酸基团，和甲苯胺蓝（ 图1），异结合磷酸基团的染料，分子分离的结合，使肌醇pyrophoshate开放研究的新途径20亚型容易检测。 页面技术的使用，净化肌醇焦磷酸酶反应产品进行outby无论是知识产权的6 K1或VIP1是一个简单，经济和可靠的方法，可以生产大量高质量的 IP 7。上面介绍的方法是不局限于简单的纯化的IP 7，但稍作修改所描述的协议可能允许不同范围的肌醇pyrophosphates净化。高等磷酸肌醇焦亚型，含有超过8个磷酸基团，可以检测到20,6作为基材使用的 IP或不同数量的 IP 6。这些肌醇pyrophosphates可以检测增加的染色过程中的长度，随后纯化（3.2节）。此外，5知识产权作为酶反应的底物的使用， 将使 PP - IP 5净化和其他含有肌醇肌醇环上的羟基pyrophosphates。 总之，这个要求不高的方法允许可靠的净化毫克量的肌醇pyrophosphates广泛使用的仪器，从而揭开了这一激动人心的研究领域的新途径。

<table border="1"><tbody><tr><th>试剂名称</th><th>公司</th><th>目录编号</th></tr><tr><td>植酸（知识产权6） </td><td> Sigma - Aldrich公司</td><td> P8810 </td></tr><tr><td>聚磷（六偏磷酸钠） </td><td> Sigma - Aldrich公司</td><td> P8510 </td></tr><tr><td> ATP - Mg 2 +的盐</td><td> Sigma - Aldrich公司</td><td> A9187 </td></tr><tr><td> OrangeG </td><td> Sigma - Aldrich公司</td><td> O3756 </td></tr><tr><td>磷酸肌酸（PCr） </td><td> Sigma - Aldrich公司</td><td> P7936 </td></tr><tr><td> CreatinePhospho激酶（CPK） </td><td> Sigma - Aldrich公司</td><td> C3755 </td></tr><tr><td> GST - VIP1 </td><td> 17 </td><td> 17 </td></tr><tr><td>他的IP6K1 </td><td> 18 </td><td> 18 </td></tr><tr><td>他的Ddp1 </td><td>可在实验室</td><td>可在实验室</td></tr><tr><td>丙烯酰胺:双丙烯酰胺为19:1（40％） </td><td> Flowgen </td><td> H16972 </td></tr><tr><td>过硫酸铵（APS） </td><td> Sigma - Aldrich公司</td><td> A9164 </td></tr><tr><td>三/硼酸/ EDTA（TBE） </td><td> Sigma - Aldrich公司</td><td> T 9060 </td></tr><tr><td> TEMED </td><td> BDH </td><td> 43083G </td></tr><tr><td>甲苯胺蓝</td><td> Sigma - Aldrich公司</td><td> 198161 </td></tr><tr><td> Speed​​Vac </td><td>基督</td><td> 100218 </td></tr><tr><td>凝胶仪器</td><td> Hoefer </td><td> SE600 </td></tr><tr><td>真空歧管</td><td>基督</td><td>阿尔法2-4 </td></tr><tr><td>真空泵</td><td>反导格莱芬伯格</td><td> 4EKF63CX </td></tr></tbody></table>

preparation of quality inositol pyrophosphates

肌醇是目前在任未修改的性质，或在更复杂的磷酸衍生物。最新的，在真核细胞中最丰富的两个pentakisphosphate肌醇（IP 5）和肌醇hexakisphosphate（植酸或IP 6）。知识产权5和IP 6都包含一个或多个焦债券1肌醇焦磷酸分子的前体。磷酸化生成的IP 6 diphoshoinositolpentakisphosphate（IP 7或PP - IP 5）和bisdiphoshoinositoltetrakisphosphate（IP 8或（PP）2 - 4的IP）。到目前为止，研究所有真核有机体肌醇pyrophosphates已被隔离。此外，负责肌醇焦磷酸合成酶的两个不同的类高度保守的整个进化2-4。 转换的IP 6激酶（知识产权6 KS）具有催化了巨大的灵活性， 知识产权 5 和 6的IP，以PP - IP 4 和 IP 7，随后，通过这些产品为底物，促进更复杂的分子的生成5,6 。近日，焦磷酸酶的第 ​​二类是确定形式的酵母蛋白VIP 1（也称为PP - IP为5 K），它能够转换成IP 6 7到IP和IP 8 7,8 。 肌醇pyrophosphates规范许多不同的细胞过程，如胰岛素的分泌，端粒长度10,11，趋化12，水泡贩运13，磷酸盐动态平衡14和HIV - 1 GAG释放15。两个行动的机制已经被提出了这一类的分子。他们可以通过allosterically与特定的蛋白质，如AKT 的 16相互作用的细胞功能的影响。此外，焦集团捐赠磷酸盐预磷酸化蛋白17。这一研究领域的巨大潜力是阻碍的肌醇pyrophosphates，这是防止许多科学家研究这些分子和这个新的翻译后修饰的商业来源的情况下。目前可用的方法，来隔离肌醇pyrophosphates需要先进的色谱仪器 18,19 。这些程序使用酸性条件下，可能会导致20肌醇焦磷酸降解，从而恢复不好。此外，繁琐的柱后脱盐程序，限制其使用专门的实验室。 在这项研究中，我们描述了一个IP 6激酶和PP - IP 5激酶反应的产品的产生，隔离和净化要求不高的方法。这种方法是可能的聚丙烯酰胺凝胶电泳（PAGE）的能力，以解决高度磷酸肌醇磷酸盐20。以下的IP 6 K1和PP - IP 为5 K为底物酶促反应的IP 6，页面用于分离，随后在水洗脱产生肌醇pyrophosphates。

Watch this Scientific Journal Video about Preparation of Quality Inositol Pyrophosphates at JoVE.com

Preparation of Quality Inositol Pyrophosphates

肌醇pyrophosphates发挥重要作用，在人类病症如癌症，糖尿病和肥胖，但行动的确切机制是一个有争议的问题。缺乏市售肌醇pyrophosphates呈现详细的研究问题。在这里，我们描述了一个简单的协议，生产和隔离毫克肌醇pyrophosphates。

编制质量肌醇Pyrophosphates

Myo-inositol is present in nature either unmodified or in more complex phosphorylated derivates. Of the latest, the two most abundant in eukaryotic cells ...

preparation-of-quality-inositol-pyrophosphates

Nanyang Technological University

Research

JoVE Journal

Biology

13.0K 次观看.  University College London. 肌醇pyrophosphates发挥重要作用，在人类病症如癌症，糖尿病和肥胖，但行动的确切机制是一个有争议的问题。缺乏市售肌醇pyrophosphates呈现详细的研究问题。在这里，我们描述了一个简单的协议，生产和隔离毫克肌醇pyrophosphates。

视频: Preparation of Quality Inositol Pyrophosphates

Horizontal Slice Preparation of the Retina

Traditionally the vertical slice and the whole-mount preparation of the retina have been used to study the function of retinal circuits. However, many of retinal neurons, such as amacrine cells, expand their dendrites horizontally, so that the morphology of the cells is supposed to be severely damaged in the vertical slices. In the whole-mount preparation, especially for patch-clamp recordings, retinal neurons in the middle layer are not easily accessible due to the extensive coverage of glial cell (Mueller cell) s endfeets. Here, we describe the novel slicing method to preserve the dendritic morphology of retinal neurons intact. The slice was made horizontally at the inner layer of the retina using a vibratome slicer after the retina was embedded in the low-temperature melting agarose gel. In this horizontal slice preparation of the retina, we studied the function of retinal neurons compared with their morphology, by using patch-clamp recording, calcium imaging technique, immunocytochemistry, and single-cell RT-PCR.

Traditionally the vertical slice and the whole-mount preparation of the retina have been used to study the function of retinal circuits. Here, we describe the novel slicing method to preserve the dendritic morphology of retinal neurons intact.

视网膜水平切片制备

Traditionally the vertical slice and the whole-mount preparation of the retina have been used to study the function of retinal circuits. However, many of ...

科研

生物学

DNA Microarrays: Sample Quality Control, Array Hybridization and Scanning

Microarray expression profiling of the nervous system provides a powerful approach to identifying gene activities in different stages of development, different physiological or pathological states, response to therapy, and, in general, any condition that is being experimentally tested1. Expression profiling of neural tissues requires isolation of high quality RNA, amplification of the isolated RNA and hybridization to DNA microarrays. In this article we describe protocols for reproducible microarray experiments from brain tumor tissue2. We will start by performing a quality control analysis of isolated RNA samples with Agilent's 2100 Bioanalyzer "lab-on-a-chip" technology. High quality RNA samples are critical for the success of any microarray experiment, and the 2100 Bioanalyzer provides a quick, quantitative measurement of the sample quality. RNA samples are then amplified and labeled by performing reverse transcription to obtain cDNA, followed by in vitro transcription in the presence of labeled nucleotides to produce labeled cRNA. By using a dual-color labeling kit, we will label our experimental sample with Cy3 and a reference sample with Cy5. Both samples will then be combined and hybridized to Agilent's 4x44 K arrays. Dual-color arrays offer the advantage of a direct comparison between two RNA samples, thereby increasing the accuracy of the measurements, in particular for small changes in expression levels, because the two RNA samples are hybridized competitively to a single microarray. The arrays will be scanned at the two corresponding wavelengths, and the ratio of Cy3 to Cy5 signal for each feature will be used as a direct measurement of the relative abundance of the corresponding mRNA. This analysis identifies genes that are differentially expressed in response to the experimental conditions being tested.

We demonstrate the use of DNA microarrays for expression profiling of the nervous system. We describe RNA quality control, sample labeling, and array hybridization and scanning.

DNA微阵列:样品的质量控制，阵列杂交和扫描

Microarray expression profiling of the nervous system provides a powerful approach to identifying gene activities in different stages of development, ...

A Noninvasive Hair Sampling Technique to Obtain High Quality DNA from Elusive Small Mammals

Noninvasive genetic sampling approaches are becoming increasingly important to study wildlife populations. A number of studies have reported using noninvasive sampling techniques to investigate population genetics and demography of wild populations1. This approach has proven to be especially useful when dealing with rare or elusive species2. While a number of these methods have been developed to sample hair, feces and other biological material from carnivores and medium-sized mammals, they have largely remained untested in elusive small mammals. In this video, we present a novel, inexpensive and noninvasive hair snare targeted at an elusive small mammal, the American pika (Ochotona princeps). We describe the general set-up of the hair snare, which consists of strips of packing tape arranged in a web-like fashion and placed along travelling routes in the pikas&#x2019; habitat. We illustrate the efficiency of the snare at collecting a large quantity of hair that can then be collected and brought back to the lab. We then demonstrate the use of the DNA IQ system (Promega) to isolate DNA and showcase the utility of this method to amplify commonly used molecular markers including nuclear microsatellites, amplified fragment length polymorphisms (AFLPs), mitochondrial sequences (800bp) as well as a molecular sexing marker. Overall, we demonstrate the utility of this novel noninvasive hair snare as a sampling technique for wildlife population biologists. We anticipate that this approach will be applicable to a variety of small mammals, opening up areas of investigation within natural populations, while minimizing impact to study organisms.

We present a noninvasive sampling approach to efficiently collect hair samples from elusive small mammals, as shown for the American pika. We demonstrate the utility of this method by extracting DNA from sampled hair and amplifying several types of molecular markers commonly used in studies of wildlife ecology and conservation.

一种无创性的发采样技术，以获得难以捉摸的小型兽类高质量的DNA

Noninvasive genetic sampling approaches are becoming increasingly important to study wildlife populations. A number of studies have reported using ...

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish water and sediment. The choice of biological indicators must be based on reliable scientific knowledge and, possibly, on the availability of standardized procedures. In this article, we present a standardized protocol that used the marine crustacean Artemia to evaluate the toxicity of chemicals and/or of marine environmental matrices. Scientists propose that the brine shrimp (Artemia) is a suitable candidate for the development of a standard bioassay for worldwide utilization. A number of papers have been published on the toxic effects of various chemicals and toxicants on brine shrimp (Artemia). The major advantage of this crustacean for toxicity studies is the overall availability of the dry cysts; these can be immediately used in testing and difficult cultivation is not demanded1,2. Cyst-based toxicity assays are cheap, continuously available, simple and reliable and are thus an important answer to routine needs of toxicity screening, for industrial monitoring requirements or for regulatory purposes3. The proposed method involves the mortality as an endpoint. The numbers of survivors were counted and percentage of deaths were calculated. Larvae were considered dead if they did not exhibit any internal or external movement during several seconds of observation4. This procedure was standardized testing a reference substance (Sodium Dodecyl Sulfate); some results are reported in this work. This article accompanies a video that describes the performance of procedural toxicity testing, showing all the steps related to the protocol.

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

This study concerns the development and standardization of a valuable methodological protocol to determine long-term (14 days) lethal toxicity exerted by chemical substances, industrial wastewater or sewage and liquid environmental samples on the saltwater crustacean, Artemia franciscana.

致命的甲壳类动物长期毒性试验卤虫franciscana</em

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish ...

Generation of High Quality Chromatin Immunoprecipitation DNA Template for High-throughput Sequencing (ChIP-seq)

ChIP-sequencing (ChIP-seq) methods directly offer whole-genome coverage, where combining chromatin immunoprecipitation (ChIP) and massively parallel sequencing can be utilized to identify the repertoire of mammalian DNA sequences bound by transcription factors in vivo. "Next-generation" genome sequencing technologies provide 1-2 orders of magnitude increase in the amount of sequence that can be cost-effectively generated over older technologies thus allowing for ChIP-seq methods to directly provide whole-genome coverage for effective profiling of mammalian protein-DNA interactions.
For successful ChIP-seq approaches, one must generate high quality ChIP DNA template to obtain the best sequencing outcomes. The description is based around experience with the protein product of the gene most strongly implicated in the pathogenesis of type 2 diabetes, namely the transcription factor transcription factor 7-like 2 (TCF7L2). This factor has also been implicated in various cancers.
Outlined is how to generate high quality ChIP DNA template derived from the colorectal carcinoma cell line, HCT116, in order to build a high-resolution map through sequencing to determine the genes bound by TCF7L2, giving further insight in to its key role in the pathogenesis of complex traits.

Generation of High Quality Chromatin Immunoprecipitation DNA Template for High-throughput Sequencing ChIP-seq

The combination of chromatin immunoprecipitation and ultra-high-throughput sequencing (ChIP-seq) can identify and map protein-DNA interactions in a given tissue or cell line. Outlined is how to generate a high quality ChIP template for subsequent sequencing, using experience with the transcription factor TCF7L2 as an example.

代高通量测序技术（ChIP-seq的高品质的染色质免疫沉淀DNA模板）

ChIP-sequencing (ChIP-seq) methods directly  offer whole-genome coverage, where combining chromatin immunoprecipitation  (ChIP) and massively parallel ...

Preparation of DNA-crosslinked Polyacrylamide Hydrogels

Mechanobiology is an emerging scientific area that addresses the critical role of physical cues in directing cell morphology and function. For example, the effect of tissue elasticity on cell function is a major area of mechanobiology research because tissue stiffness modulates with disease, development, and injury. Static tissue-mimicking materials, or materials that cannot alter stiffness once cells are plated, are predominately used to investigate the effects of tissue stiffness on cell functions. While information gathered from static studies is valuable, these studies are not indicative of the dynamic nature of the cellular microenvironment in vivo. To better address the effects of dynamic stiffness on cell function, we developed a DNA-crosslinked polyacrylamide hydrogel system (DNA gels). Unlike other dynamic substrates, DNA gels have the ability to decrease or increase in stiffness after fabrication without stimuli. DNA gels consist of DNA crosslinks that are polymerized into a polyacrylamide backbone. Adding and removing crosslinks via delivery of single-stranded DNA allows temporal, spatial, and reversible control of gel elasticity. We have shown in previous reports that dynamic modulation of DNA gel elasticity influences fibroblast and neuron behavior. In this report and video, we provide a schematic that describes the DNA gel crosslinking mechanisms and step-by-step instructions on the preparation DNA gels.

Our laboratory has developed DNA-crosslinked polyacrylamide hydrogels, a dynamic hydrogel system, to better understand the effects of modulating tissue stiffness on cell function. Here, we provide schematics, descriptions, and protocols to prepare these hydrogels.

DNA交联聚丙烯酰胺水凝胶的制备

Mechanobiology is an emerging scientific area that addresses the critical role of physical cues in directing cell morphology and function. For example, ...

Do's and Don'ts of Cryo-electron Microscopy: A Primer on Sample Preparation and High Quality Data Collection for Macromolecular 3D Reconstruction

Cryo-electron microscopy (cryoEM) entails flash-freezing a thin layer of sample on a support, and then visualizing the sample in its frozen hydrated state by transmission electron microscopy (TEM). This can be achieved with very low quantity of protein and in the buffer of choice, without the use of any stain, which is very useful to determine structure-function correlations of macromolecules. When combined with single-particle image processing, the technique has found widespread usefulness for 3D structural determination of purified macromolecules. 
The protocol presented here explains how to perform cryoEM and examines the causes of most commonly encountered problems for rational troubleshooting; following all these steps should lead to acquisition of high quality cryoEM images. The technique requires access to the electron microscope instrument and to a vitrification device. Knowledge of the 3D reconstruction concepts and software is also needed for computerized image processing. Importantly, high quality results depend on finding the right purification conditions leading to a uniform population of structurally intact macromolecules. 
The ability of cryoEM to visualize macromolecules combined with the versatility of single particle image processing has proven very successful for structural determination of large proteins and macromolecular machines in their near-native state, identification of their multiple components by 3D difference mapping, and creation of pseudo-atomic structures by docking of x-ray structures. The relentless development of cryoEM instrumentation and image processing techniques for the last 30 years has resulted in the possibility to generate de novo 3D reconstructions at atomic resolution level.

This paper describes the cryo-electron microscopy methodology, which is used to obtain high quality microscopic images of macromolecules in their near-native state. The method yields images suitable for further computerized processing using the single particle approach, devised to generate the 3D reconstruction of a macromolecule.

该做什么和不该做什么低温电子显微镜的:入门样品制备和高质量的数据采集为大分子三维重建

Cryo-electron microscopy (cryoEM) entails flash-freezing a thin layer of sample on a support, and then visualizing the sample in its frozen hydrated state ...

A Web Tool for Generating High Quality Machine-readable Biological Pathways

PathWhiz is a web server built to facilitate the creation of colorful, interactive, visually pleasing pathway diagrams that are rich in biological information. The pathways generated by this online application are machine-readable and fully compatible with essentially all web-browsers and computer operating systems. It uses a specially developed, web-enabled pathway drawing interface that permits the selection and placement of different combinations of pre-drawn biological or biochemical entities to depict reactions, interactions, transport processes and binding events. This palette of entities consists of chemical compounds, proteins, nucleic acids, cellular membranes, subcellular structures, tissues, and organs. All of the visual elements in it can be interactively adjusted and customized. Furthermore, because this tool is a web server, all pathways and pathway elements are publicly accessible. This kind of pathway &#34;crowd sourcing&#34; means that PathWhiz already contains a large and rapidly growing collection of previously drawn pathways and pathway elements. Here we describe a protocol for the quick and easy creation of new pathways and the alteration of existing pathways. To further facilitate pathway editing and creation, the tool contains replication and propagation functions. The replication function allows existing pathways to be used as templates to create or edit new pathways. The propagation function allows one to take an existing pathway and automatically propagate it across different species. Pathways created with this tool can be &#34;re-styled&#34; into different formats (KEGG-like or text-book like), colored with different backgrounds, exported to BioPAX, SBGN-ML, SBML, or PWML data exchange formats, and downloaded as PNG or SVG images. The pathways can easily be incorporated into online databases, integrated into presentations, posters or publications, or used exclusively for online visualization and exploration. This protocol has been successfully applied to generate over 2,000 pathway diagrams, which are now found in many online databases including HMDB, DrugBank, SMPDB, and ECMDB.

PathWhiz is a comprehensive, online pathway drawing tool for generating biochemical and biological pathways. It uses publicly accessible databases and easily expandable palettes consisting of pre-drawn pathway components. This protocol describes how to easily build new pathways, replicate and edit existing pathways, and propagate previously drawn pathways to different organisms.

一个网络工具，用以生成高质量机器可读的生物学途径

PathWhiz is a web server built to facilitate the creation of colorful, interactive, visually pleasing pathway diagrams that are rich in biological ...

Sperm Collection of Differential Quality Using Density Gradient Centrifugation

In sexual reproduction, a male gamete or sperm cell fuses with a female gamete to bring about fertilization. However, a large number of sperm cells with fertilizing ability are required to interact with a female gamete to ensure fertilization. As such, the fertilizing ability of individual sperm cells is critical for successful reproduction. Density gradient centrifugation has been utilized for several decades as a reproducible, fast, efficient, effective and extremely adaptable method to collect only high-quality sperm to be used in assisted reproductive technology. The protocols we described herein focus on the utilization of the discontinuous Percoll density gradient centrifugation (PDGC) technique to isolate three distinct populations of rooster sperm by their quality. We were able to collect low-, medium- and high-quality sperm. We also describe reproducible protocols that entail determining fertility potential of sperm by assessing their viability, mobility and penetrability. Collection of sperm by their quality using PDGC technique would be useful to accurately and thoroughly characterize sperm with differential fertility potential.

In this paper, we aim to describe the performance of the density gradient centrifugation technique and its application in sperm physiology research.

密度梯度离心法测定微分品质的精子

In sexual reproduction, a male gamete or sperm cell fuses with a female gamete to bring about fertilization. However, a large number of sperm cells with ...

Field Identification of Matricaria chamomilla using a Portable qPCR System

Quality control in botanical products begins with the raw material supply. Traditionally, botanical identification is performed through morphological assessment and chemical analytical methods. However, the lack of availability of botanists, especially in recent years, coupled with the need to enhance quality control to combat the stresses on the supply chain brought by increasing consumer demand and climate change, necessitates alternative approaches. The goal of this protocol is to facilitate botanical species identification using a portable qPCR system on the field or in any setting, where access to laboratory equipment and expertise is limited. Target DNA is amplified using dye-based qPCR, with DNA extracted from botanical reference materials serving as a positive control. The target DNA is identified by its specific amplification and matching its melting peak against the positive control. A detailed description of the steps and parameters, from hands-on field sample collection, to DNA extraction, PCR amplification, followed by data interpretation, has been included to ensure that readers can replicate this protocol. The results produced align with traditional laboratory botanical identification methods. The protocol is easy to perform and cost-effective, enabling quality testing on raw materials as close to the point of origin of the supply chain as possible.

Field Identification of Matricaria chamomilla using a Portable qPCR System

Presented here is a protocol for field identification of Matricaria chamomilla using a portable qPCR system. This easy-to-perform protocol is ideal as a method to confirm the identity of a botanical species at locations where access to laboratory equipment and expertise is limited, such as farms and warehouses.

使用便携式 qPCR 系统对 马特里卡里亚查莫米拉进行现场识别

Quality control in botanical products begins with the raw material supply. Traditionally, botanical identification is performed through morphological ...