这项工作是由加拿大卫生研究院研究院（CIHR），国家科学与工程研究理事会（NSERC）和CIHR培训资助奖学金（LF）的支持。我们感谢教授杰拉德D.赖特（麦克马斯特大学，加拿大）（6）为AAC - II表达质粒。

1。准备库存解决方案<ol><li>净化大分子的兴趣。 （在这种情况下，氨基糖苷类的N - 6&#39; -乙酰基- II（AAC6&#39;二），是孤立的报道13。） </li><li>准备4公升的透析液。 （在这种情况下，我们使用25毫米4 - （2 - 羟乙基）- 1 - piperazineethanesulfonic酸（HEPES，MW​​ 238.3克/摩尔），含有2毫米乙二胺四乙酸（EDTA，292.2兆瓦），在pH值7.5。） </li><li>透析的AAC（6&#39;）- II的样品在400微米（5毫升），用于必须在透析透析缓冲液（3 × 1.3升）的蛋白质。保持最后的透析液冲洗机和稀释样品。 </li><li>最终的透析液，通过0.45微米的纤维滤纸过滤，并保存在4 ° C将被称为"运行缓冲" </li><li>通过0.2微米注射器的过滤器已运行缓冲液彻底冲洗过滤蛋白质溶液。测量使用标准的检测（布拉德福德，洛瑞等）或紫外吸收的蛋白质终浓度。商店的蛋白质，以最大限度地长期稳定。 （AAC（6&#39;）- II，这意味着存储在4 ° C的AAC（6&#39;）- II不保留冷冻和解冻后的活动）。 </li><li>准备运行缓冲液溶解在4.0毫克200μL25毫米乙酰辅酶A的股票的解决方案（AcCoA，配体，809.57兆瓦）。冻结在-78 ° C，直到准备使用。 </li><li>所有的蛋白质与配体样本必须来自同一只股票的解决方案，以最大限度地减少随机抽样样本浓度波动。一个横跨整个变量- C数据集的蛋白浓度修正系数调整分析 10 。 </li></ol> 2。准备美国国际贸易委员会样本<ol><li>酶液稀释到64 C值与终体积为2 mL。 （AAC（6&#39;）- II的情况下，这相当于到192微米。） </li><li>快速解冻，在冰水中的配体（AcCoA）原液。 </li><li>准备一个AcCoA解决方案的0.5 mL的10倍以上的浓缩，然后在对蛋白质的结合位点的数量，在这种情况下，4毫米，420μL运行缓冲液80μLAcCoA原液稀释。转移到一个吸管灌浆管。快速返回AcCoA原液至-78 ° C。 </li><li>德加的蛋白质和真空条件下的解决方案，在温度为1 5分钟℃以下所需的运行温度（19℃）。 </li></ol> 3。设立注射器14 <ol><li>作为持有人在同一高度，直到预安装的注射器钳，通过注射器持有插入注射器。 </li><li>第二注射器钳过的注射器，注射器支架的底部，直到它坚决反对按饲料。轻轻地拧紧钳提供0.050"球点六角驱动。 </li><li>放入注射器吸液管人持有。 </li><li>轻轻地滑入注射器吸液管注入。确保柱塞头孔的注射器直接送入。一旦完全插入，拧到吸管注射器注射器持有的锁圈。 </li></ol> 4。装入样品池14 <ol><li>最低限度的运行缓冲液50毫升清洗样品池，并删除任何剩余的液体，用长针刺2.5毫升的玻璃注射器。 </li><li>慢慢地画出一个进入清洁和干燥的长针2.5毫升注射器的蛋白质样品溶液1.8毫升的最低。要小心，不要采取任何气泡。 </li><li>小心地插入样品池针，轻轻触摸单元格的底部。提高尖稍（〜1毫米），轻轻注入细胞的AAC（6&#39;）- II解决方案，直到多余的液体样品池的顶部以上可见。 </li><li>同时确保在溢出的液体仍然慢​​慢提高约1厘米的针。迅速退出，并注入少量的溶液（〜0.25毫升）样品池，以消除任何被困气泡。 </li><li>删除所有的解决方案溢出。这是通过轻轻滑动沿溢出方针进样细胞。注射器尖端将达到一个窗台，这是希望的解决方案运行的高度。删除位于这条线以上的所有液体。 </li></ol> 5。装入注射器，并开始运行 14 <ol><li>将填写端口塑料管装载注射器。 </li><li>下柱塞提示填写端口。 </li><li>在吸管持有人的底部放置吸管灌浆管AcCoA解决方案。注射器尖不应该接触的灌装管的底部。 </li><li>慢慢地绘制到注射器的解决方案，直到少量进入装载注射器管。 </li><li>降低柱塞头，然后单击"关闭填写端口"按钮，关闭填写端口。清除清除&gt;填充按钮，点击3次，以消除任何气泡和填充，很可能会成为被困在装载。 </li><li>从吸管人删除灌浆管，轻轻擦拭注射器的尖端。 </li><li>以吸管组装成的样品池，并轻​​轻地放下注射器。进展缓慢的注射器可以很容易地弯曲，所以需要非常谨慎。确保完全按下锁圈的基础上，插入注射器。 </li><li>设置所需的运行温度（29 ° C在这种情况下），并选择一个参考功率，略高于预期（在这种情况下20μcal/sec）的流的最大注射热。 </li><li>编程所需的注射量和延误。 （在这种情况下，28名注射就业。第一次注射容积2μL与60秒的延时，所有后续注射10μL330章第延迟卷。） </li></ol> 6。随后的运行<ol><li>重复步骤2-5在随后的运行，同时降低AAC（6&#39;）- II和AcCoA浓度的2,4,8,16倍，而32。 </li></ol> 7。数据分析<ol><li>使用全球适合所有的等温线到一个单一的绑定参数设置一个合适的程序，以前10描述。 </li></ol> 8。代表性的成果具有代表性的数据，如图1所示。等温线的形状应与浓度的不同而有所差异。更清晰的转换，预计较高的C -值（即较高的蛋白质和配体浓度）（图2）。 AAC（6&#39;）- II的情况下，两个网站的序列模型，给出了一个更适合于描述两套相同的独立网站，具有可调节的化学计量。 <img src="/files/ftp_upload/2529/2529fig1.jpg" alt="图1" /> AcCoA滴定（3.86毫米）制作成AAC（6&#39;）- II（192μM） 图1等温线。一）原国际贸易中心跟踪。 b）用于确定一个适合2现场连​​续（绑定参数（正方形）的综合值 - ）。 <img src="/files/ftp_upload/2529/2529fig2.jpg" alt="图2" /> 图2。贸易中心AcCoA等温线在不同浓度的滴定成AAC（6&#39;）- II。适合2集站点的独立模式（虚线洋红）和2现场连续模型（实心蓝色）的实验数据（空心圆）。 2现场sequentional模式显然会带来更好的整体协议。浓度分别为A）6微米，0.25毫米，二）12微米，0.25毫米，C）24微米，0.5 mm，深）48μM，1.0毫米，E），96μM，1.9毫米，和F）196μM， 3.86毫米，为AAC（6&#39;）- II和AcCoA分别。

<ol>
	<li>Cliff, M. J., Ladbury, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+survey+of+the+year+2002+literature+on+applications+of+isothermal+titration+calorimetry.">A survey of the year 2002 literature on applications of isothermal titration calorimetry.</a> Journal of Molecular Recognition. 16, 383-391 (2003).</li><li>Cliff, M. J., Gutierrez, A., Ladbury, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+survey+of+the+year+2003+literature+on+applications+of+isothermal+titration+calorimetry.">A survey of the year 2003 literature on applications of isothermal titration calorimetry.</a> Journal of Molecular Recognition. 17, 513-523 (2004).</li><li>Ababou, A., Ladbury, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Survey+of+the+year+2004:+literature+on+applications+of+isothermal+titration+calorimetry.">Survey of the year 2004: literature on applications of isothermal titration calorimetry.</a> Journal of Molecular Recognition. 19, 79-89 (2006).</li><li>Ababou, A., Ladbury, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Survey+of+the+year+2005:+literature+on+applications+of+isothermal+titration+calorimetry.">Survey of the year 2005: literature on applications of isothermal titration calorimetry.</a> Journal of Molecular Recognition. 20, 4-14 (2007).</li><li>Okhrimenko, O. k. s. a. n. a., J, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+survey+of+the+year+2006+literature+on+applications+of+isothermal+titration+calorimetry.">A survey of the year 2006 literature on applications of isothermal titration calorimetry.</a> Journal of Molecular Recognition. 21, 1-19 (2008).</li><li>Bjelic, S., Jelesarov, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+survey+of+the+year+2007+literature+on+applications+of+isothermal+titration+calorimetry.">A survey of the year 2007 literature on applications of isothermal titration calorimetry.</a> Journal of Molecular Recognition. 21, 289-312 (2008).</li><li>Leavitt, S., Freire, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+measurement+of+protein+binding+energetics+by+isothermal+titration+calorimetry.">Direct measurement of protein binding energetics by isothermal titration calorimetry.</a> Current Opinion in Structural Biology. 11, 560-566 (2001).</li><li>Wiseman, T., Williston, S., Brandts, J. F., Lin, L. -. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+measurement+of+binding+constants+and+heats+of+binding+using+a+new+titration+calorimeter.">Rapid measurement of binding constants and heats of binding using a new titration calorimeter.</a> Analytical Biochemistry. 179, 131-137 (1989).</li><li>Capaldi, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+X-Ray+Structure+of+Zebrafish+(Danio+rerio)+Ileal+Bile+Acid-Binding+Protein+Reveals+the+Presence+of+Binding+Sites+on+the+Surface+of+the+Protein+Molecule.">The X-Ray Structure of Zebrafish (Danio rerio) Ileal Bile Acid-Binding Protein Reveals the Presence of Binding Sites on the Surface of the Protein Molecule.</a> Journal of Molecular Biology. 385, 99-116 (2009).</li><li>Freiburger, L. A., Auclair, K., Mittermaier, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elucidating+Protein+Binding+Mechanisms+by+Variable-c+ITC.">Elucidating Protein Binding Mechanisms by Variable-c ITC.</a> ChemBioChem. 10, 2871-2873 (2009).</li><li>Wybenga-Groot, L. E., Draker, K. -. a., Wright, G. D., Berghuis, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystal+structure+of+an+aminoglycoside+6'-N-acetyltransferase:+defining+the+GCN5-related+N-acetyltransferase+superfamily+fold.">Crystal structure of an aminoglycoside 6'-N-acetyltransferase: defining the GCN5-related N-acetyltransferase superfamily fold.</a> Structure. 7, 497-507 (1999).</li><li>Draker, K., Northrop, D. B., Wright, G. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinetic+Mechanism+of+the+GCN5-Related+Chromosomal+Aminoglycoside+Acetyltransferase+AAC(6')-Ii+from+Enterococcus+faecium:+Evidence+of+Dimer+Subunit+Cooperativity.">Kinetic Mechanism of the GCN5-Related Chromosomal Aminoglycoside Acetyltransferase AAC(6')-Ii from Enterococcus faecium: Evidence of Dimer Subunit Cooperativity.</a> Biochemistry. 42, 6565-6574 (2003).</li><li>Wright, G. D., Ladak, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overexpression+and+characterization+of+the+chromosomal+aminoglycoside+6'-N-acetyltransferase+from+Enterococcus+faecium.">Overexpression and characterization of the chromosomal aminoglycoside 6'-N-acetyltransferase from Enterococcus faecium.</a> Antimicrob. Agents Chemother. 41, 956-960 (1997).</li><li>MicroCal. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> ITC Data Analysis in Origin. , (2004).</li></ol>

这个变量- C管件的分析部分以前已经10详细描述。在这里，我们报告收集适合这种方法的变量- C的数据集的实际问题。重要的是，所有的蛋白质与配体样本是从同一只股票方案的制定。因此重要的是，准备充足的库存解决方案，初步完成了整个系列实验。这将确保比例的AAC（6&#39;）- II和AcCoA是不变的，所有的实验中，并降低浓度样本，以抽样方式随机波动。 在这种情?...

<!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>Acetyl coenzyme A (AcCoA)</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>A2056</td>
<td>&nbsp;</td>
<tr>
<td>4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>7365-45-9</td>
<td>&nbsp;</td>
<tr>
<td>ethylenediaminetetraacetic acid (EDTA)</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>431788</td>
<td>&nbsp;</td>
<tr>
<td>Spectra/Por 2 Dialysis Tubing</td>
<td>&nbsp;</td>
<td>Spectrum Labs</td>
<td>132678</td>
<td>&nbsp;</td>
<tr>
<td>Sterile Syringe Filter (0.2 &mu;m)</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>281445-477</td>
<td>&nbsp;</td>
<tr>
<td>Cellulos Nitrate Membrane Filters (0.45 &mu;m)</td>
<td>&nbsp;</td>
<td>Whatman</td>
<td>7184-004</td>
<td>&nbsp;</td>
<tr>
<td>VP-ITC</td>
<td>&nbsp;</td>
<td>MicroCal</td>
<td>VP-ITC</td>
<td>Microcalorimeter used for measurements</td>
</tr>
<tr>
<td>ThermoVac</td>
<td>&nbsp;</td>
<td>MicroCal</td>
<td>USB Thermo Vac</td>
<td>Temperature Controlled Degassing Station</td>
</tr>		</tbody>
		</table>
		</div>

collecting variable-concentration isothermal titration calorimetry datasets in order to determine binding mechanisms

等温滴定量热（ITC）是常用的确定与主机高分子配体的结合相关的热力学参数。 ITC主机/配体的相互作用研究一些较常见的光谱方法的优点。例如，两个组件交互时释放或吸收的热量是直接测量，不需要任何外源性的记者。因此，结合焓和协会常数（Ka）直接获得从国际贸易中心的数据，可用于计算熵的贡献。此外，等温线的形状是依赖于C值和所涉及的机理模型。 C -值被定义为C = N [P] TKA，[P] T是蛋白浓度，n是主机内配体结合位点的数目。在许多情况下，为一个给定的配体的多个结合位点非等效ITC允许每一个人的结合位点的热力学约束力参数的表征。然而，这需要使用正确的绑定模型。这种选择可能会产生问题，如果不同型号，可容纳相同的实验数据。我们曾表明，这个问题是可以规避在几个C -值进行实验。同时适合不同的模型在不同的C -值获得的多个等温线。正确的模式是未来整个变量- C数据集的拟合善良的基础上确定。这个过程是应用氨基糖甙类抗生素导致耐药酶氨基糖苷类的N - 6&#39; -乙酰基转移酶- II（AAC（6&#39;）- II）。虽然我们的方法是适用于任何系统，更好地证明这一战略的必要性与高分子配体变构或协同系统，和不同的结合模式提供相同的数据基本上是相同的适合。据我们所知，有没有市售的这类系统。 AAC（6&#39;）- II，是一个同源二聚体，含有两个活性部位，呈现出两个亚基之间的协同。然而，在一个单一的C -价值得到国际贸易中心的数据，可以适合同样至少有两个不同型号的两集网站独立模型和两站点顺序（合作）模式。通过不同的C值，如上面所述，它是建立正确的绑定模型的AAC（6&#39;）- II是两个站点的顺序的绑定模型。在这里，我们描述的，必须在执行国际贸易中心实验时所采取的的，为了获得适合变量- C分析数据集的步骤。

Watch this Scientific Journal Video about Collecting Variable-concentration Isothermal Titration Calorimetry Datasets in Order to Determine Binding Mechanisms at JoVE.com

Collecting Variable-concentration Isothermal Titration Calorimetry Datasets in Order to Determine Binding Mechanisms

ITC是一个功能强大的工具，为研究其主机的一个配体的结合。然而，在复杂的系统的几种模式可能适合同样的数据。这里介绍的方法，提供了一种手段来澄清合适的绑定模型和复杂的系统，提取相应的热力学参数。

收集可变浓度等温滴定量热数据集，以确定约束机制

Isothermal titration calorimetry (ITC) is commonly used to determine the thermodynamic parameters associated with the binding of a ligand to a host ...

collecting-variable-concentration-isothermal-titration-calorimetry

Research

JoVE Journal

Biology

12.7K 次观看.  McGill University. ITC是一个功能强大的工具，为研究其主机的一个配体的结合。然而，在复杂的系统的几种模式可能适合同样的数据。这里介绍的方法，提供了一种手段来澄清合适的绑定模型和复杂的系统，提取相应的热力学参数。

视频: Collecting Variable-concentration Isothermal Titration Calorimetry Datasets in Order to Determine Binding Mechanisms

Investigating the Immunological Mechanisms Underlying Organ Transplant Rejection

调查的基本免疫机制，器官移植排斥反应

科研

生物学

Collecting and Measuring Nociceptive and Inflammatory Mediators in Surgical Wounds 

We describe a methodology by which we are able to collect and measure inflammatory and nociceptive biochemical mediators at the surgical wound site. Collecting site-specific biochemical markers allows us to evaluate the relationship between surgical wound and serum levels; determine any associations between mediator release, pain and analgesic consumption; and evaluate the effect of systemic and peripheral drug administration on surgical wound biochemistry.
This methodology has been applied to healthy women undergoing elective cesarean delivery with spinal anesthesia. Wound exudate and serum mediators, in conjunction with pain scores and analgesics consumption were measured at 1, 6, 24, and 48 hours post-cesarean delivery. Biochemical mediators that were detected included IL-1&beta;, IL-2, IL-4, IL-6, IL-7, IL-8, IL-10, IL-12, IL-13, IL-17, TNF&alpha;, INF&gamma;, G-CSF, GM-CSF, MCP-1 and MIP-1&beta;, nerve growth factor (NGF), prostaglandin E2 (PG-E2) and substance P. We found no correlations between wound and serum cytokines concentrations or time-release profiles (J Pain. 2008 Jul 9(7):650-7). This article describes and demonstrates the feasibility of collecting and assaying nociceptive and inflammatory mediators in surgical wounds at specific time points. The lack of significant correlations between serum and wound levels shows the importance of determining site-specific release if surgical wounds and localized pathologies are to be studied.

Collecting and Measuring Nociceptive and Inflammatory Mediators in Surgical Wounds

A technique to collect and measure surgical wound biochemical mediators at specific time points.

在手术伤口的痛觉及炎症介质的收集和测量

We describe a methodology by which we are able to collect and measure inflammatory and nociceptive biochemical mediators at the surgical wound site. ...

Use of the Protease Fluorescent Detection Kit to Determine Protease Activity

The Protease Fluorescent Detection Kit provides ready-to-use reagents for detecting the presence of protease activity. This simple assay to detect protease activity uses casein labeled with fluorescein isothiocyanate (FITC) as the substrate.
Protease activity results in the cleavage of the FITC-labeled casein substrate into smaller fragments, which do not precipitate under acidic conditions. After incubation of the protease sample and substrate, the reaction is acidified with the addition of trichloroacetic acid (TCA). The mixture is then centrifuged with the undigested substrate forming a pellet and the smaller, acid soluble fragments remaining in solution. The supernatant is neutralized and the fluorescence of the FITC-labeled fragments is measured.
The described kit procedure detects the trypsin protease control at a concentration of approximately 0.5 &mu;g/ml (5 ng of trypsin added to the assay). This sensitivity can be increased with a longer incubation time, up to 24 hours. The assay is performed in microcentrifuge tubes and procedures are provided for fluorescence detection using either cuvettes or multiwell plates.

The Protease Fluorescent Detection Kit is designed for the measurement of protease activity using fluorometry. It is also suitable for detection of trace amounts of protease contamination. The method is based on the proteolytic hydroysis of a proprietary formulation of a FITC-labeled casein substrate.

蛋白酶荧光检测试剂盒，用它来确定蛋白酶活性

The Protease Fluorescent Detection Kit provides ready-to-use reagents for detecting the presence of protease activity. This simple assay to detect ...

PAR-CliP - A Method to Identify Transcriptome-wide the Binding Sites of RNA Binding Proteins

RNA transcripts are subjected to post-transcriptional gene regulation by interacting with hundreds of RNA-binding proteins (RBPs) and microRNA-containing ribonucleoprotein complexes (miRNPs) that are often expressed in a cell-type dependently. To understand how the interplay of these RNA-binding factors affects the regulation of individual transcripts, high resolution maps of in vivo protein-RNA interactions are necessary1.
A combination of genetic, biochemical and computational approaches are typically applied to identify RNA-RBP or RNA-RNP interactions. Microarray profiling of RNAs associated with immunopurified RBPs (RIP-Chip)2 defines targets at a transcriptome level, but its application is limited to the characterization of kinetically stable interactions and only in rare cases3,4 allows to identify the RBP recognition element (RRE) within the long target RNA. More direct RBP target site information is obtained by combining in vivo UV crosslinking5,6 with immunoprecipitation7-9 followed by the isolation of crosslinked RNA segments and cDNA sequencing (CLIP)10. CLIP was used to identify targets of a number of RBPs11-17. However, CLIP is limited by the low efficiency of UV 254 nm RNA-protein crosslinking, and the location of the crosslink is not readily identifiable within the sequenced crosslinked fragments, making it difficult to separate UV-crosslinked target RNA segments from background non-crosslinked RNA fragments also present in the sample.
We developed a powerful cell-based crosslinking approach to determine at high resolution and transcriptome-wide the binding sites of cellular RBPs and miRNPs that we term PAR-CliP (Photoactivatable-Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation) (see Fig. 1A for an outline of the method). The method relies on the incorporation of photoreactive ribonucleoside analogs, such as 4-thiouridine (4-SU) and 6-thioguanosine (6-SG) into nascent RNA transcripts by living cells. Irradiation of the cells by UV light of 365 nm induces efficient crosslinking of photoreactive nucleoside-labeled cellular RNAs to interacting RBPs. Immunoprecipitation of the RBP of interest is followed by isolation of the crosslinked and coimmunoprecipitated RNA. The isolated RNA is converted into a cDNA library and deep sequenced using Solexa technology. One characteristic feature of cDNA libraries prepared by PAR-CliP is that the precise position of crosslinking can be identified by mutations residing in the sequenced cDNA. When using 4-SU, crosslinked sequences thymidine to cytidine transition, whereas using 6-SG results in guanosine to adenosine mutations. The presence of the mutations in crosslinked sequences makes it possible to separate them from the background of sequences derived from abundant cellular RNAs.
Application of the method to a number of diverse RNA binding proteins was reported in Hafner et al.18

RNA transcripts are subject to extensive posttranscriptional regulation that is mediated by a multitude of trans-acting RNA-binding proteins (RBPs). Here we present a generalizable method to identify precisely and on a transcriptome-wide scale the RNA binding sites of RBPs.

PAR剪辑 - 一个方法来确定全转录组学，RNA结合蛋白的结合位点

RNA transcripts are subjected to post-transcriptional gene regulation by interacting with hundreds of RNA-binding proteins (RBPs) and microRNA-containing ...

Mouse Embryonic Lung Culture, A System to Evaluate the Molecular Mechanisms of Branching

Lung primordial specification as well as branching morphogenesis, and the formation of various pulmonary cell lineages requires a specific interaction of the lung endoderm with its surrounding mesenchyme and mesothelium. Lung mesenchyme has been shown to be the source of inductive signals for lung branching morphogenesis. Epithelial-mesenchymal-mesothelial interactions are also critical to embryonic lung morphogenesis. Early embryonic lung organ culture is a very useful system to study epithelial-mesenchymal interactions. Both epithelial and mesenchymal morphogenesis proceeds under specific conditions that can be readily manipulated in this system (in the absence of maternal influence and blood flow). More importantly this technique can be readily done in a serumless, chemically defined culture media. Gain and loss of function can be achieved using expressed proteins, recombinant viral vectors and/or analysis of transgenic mouse strains, antisense RNA, as well as RNA interference gene knockdown.

Early embryonic lung organ culture is a very useful system to study epithelial-mesenchymal interactions. Both epithelial and mesenchymal morphogenesis proceeds under specific conditions that can be readily manipulated in this system.

小鼠胚胎的龙文化，一个系统来评价分枝的分子机制

Lung primordial specification as well as branching morphogenesis, and the formation of various pulmonary cell lineages requires a specific interaction of ...

Isothermal Titration Calorimetry for Measuring Macromolecule-Ligand Affinity

Isothermal titration calorimetry (ITC) is a useful tool for understanding the complete thermodynamic picture of a binding reaction. In biological sciences, macromolecular interactions are essential in understanding the machinery of the cell. Experimental conditions, such as buffer and temperature, can be tailored to the particular binding system being studied. However, careful planning is needed since certain ligand and macromolecule concentration ranges are necessary to obtain useful data. Concentrations of the macromolecule and ligand need to be accurately determined for reliable results. Care also needs to be taken when preparing the samples as impurities can significantly affect the experiment. When ITC experiments, along with controls, are performed properly, useful binding information, such as the stoichiometry, affinity and enthalpy, are obtained. By running additional experiments under different buffer or temperature conditions, more detailed information can be obtained about the system. A protocol for the basic setup of an ITC experiment is given.

A general protocol for the use of isothermal titration calorimetry to monitor the binding thermodynamics for biological systems with moderate binding affinities is presented.

用于测量高分子配体亲和等温滴定量热

Isothermal titration calorimetry (ITC) is a useful tool for understanding the complete thermodynamic picture of a binding reaction.  In biological ...

Culturing Primary Rat Inner Medullary Collecting Duct Cells

Arginine-vasopressin (AVP) facilitates water reabsorption by renal collecting duct principal cells and thereby fine-tunes body water homeostasis. AVP binds to vasopressin V2 receptors (V2R) on the surface of the cells and thereby induces synthesis of cAMP. This stimulates cellular signaling processes leading to changes in the phosphorylation of the water channel aquaporin-2 (AQP2). Protein kinase A phoshorylates AQP2 and thereby triggers the translocation of AQP2 from intracellular vesicles into the plasma membrane facilitating water reabsorption from primary urine. Aberrations of AVP release from the pituitary or AVP-activated signaling in principal cells can cause central or nephrogenic diabetes insipidus, respectively; an elevated blood plasma AVP level is associated with cardiovascular diseases such as chronic heart failure and the syndrome of inappropriate antidiuretic hormone secretion.
Here, we present a protocol for cultivation of primary rat inner medullary collecting duct (IMCD) cells, which express V2R and AQP2 endogenously. The cells are suitable for elucidating molecular mechanisms underlying the control of AQP2 and thus to discover novel drug targets for the treatment of diseases associated with dysregulation of AVP-mediated water reabsorption. IMCD cells are obtained from rat renal inner medullae and are used for experiments six to eight days after seeding. IMCD cells can be cultured in regular cell culture dishes, flasks and micro-titer plates of different formats, the procedure only requires a few hours, and is appropriate for standard cell culture laboratories.

Arginine-vasopressin (AVP) controls fine-tuning of body water homeostasis through facilitating water reabsorption by renal principal cells. Here, we present a protocol for the cultivation of primary rat inner medullary collecting duct cells suitable for the elucidation of molecular mechanisms underlying AVP-mediated water reabsorption.

原代培养大鼠内髓集合管细胞

Arginine-vasopressin (AVP) facilitates water reabsorption by renal collecting duct principal cells and thereby fine-tunes body water homeostasis. AVP ...

Biochemical Titration of Glycogen In vitro

Glycogen is the main energetic polymer of glucose in vertebrate animals and plays a crucial role in whole body metabolism as well as in cellular metabolism. Many methods to detect glycogen already exist but only a few are quantitative. We describe here a method using the Abcam Glycogen assay kit, which is based on specific degradation of glycogen to glucose by glucoamylase. Glucose is then specifically oxidized to a product that reacts with the OxiRed probe to produce fluorescence. Titration is accurate, sensitive and can be achieved on cell extracts or tissue sections. However, in contrast to other techniques, it does not give information about the distribution of glycogen in the cell. As an example of this technique, we describe here the titration of glycogen in two cell lines, Chinese hamster lung fibroblast CCL39 and human colon carcinoma LS174, incubated in normoxia (21% O2) versus hypoxia (1% O2). We hypothesized that hypoxia is a signal that prepares cells to synthesize and store glycogen in order to survive1.

Biochemical Titration of Glycogen In vitro

We describe here an accurate, reproducible and convenient biochemical method for titration of glycogen in vitro. This technique uses the Abcam Glycogen assay kit and is based on successive hydrolysis of glycogen to glucose and glucose titration by fluorescence.

糖原生化滴定<em&gt;在体外</em

Glycogen is the main energetic polymer of glucose in vertebrate animals and plays a crucial role in whole body metabolism as well as in cellular ...

Isolation of mRNAs Associated with Yeast Mitochondria to Study Mechanisms of Localized Translation

Most of mitochondrial proteins are encoded in the nucleus and need to be imported into the organelle. Import may occur while the protein is synthesized near the mitochondria. Support for this possibility is derived from recent studies, in which many mRNAs encoding mitochondrial proteins were shown to be localized to the mitochondria vicinity. Together with earlier demonstrations of ribosomes&#8217; association with the outer membrane, these results suggest a localized translation process. Such localized translation may improve import efficiency, provide unique regulation sites and minimize cases of ectopic expression. Diverse methods have been used to characterize the factors and elements that mediate localized translation. Standard among these is subcellular fractionation by differential centrifugation. This protocol has the advantage of isolation of mRNAs, ribosomes and proteins in a single procedure. These can then be characterized by various molecular and biochemical methods. Furthermore, transcriptomics and proteomics methods can be applied to the resulting material, thereby allow genome-wide insights. The utilization of yeast as a model organism for such studies has the advantages of speed, costs and simplicity. Furthermore, the advanced genetic tools and available deletion strains facilitate verification of candidate factors.

Many mRNAs encoding mitochondrial proteins are associated with the mitochondria outer membrane. We describe a subcellular fractionation procedure aimed at isolation of yeast mitochondria with its associated mRNAs and ribosomes. This protocol can be applied to cells grown under diverse conditions&#160;in order to reveal mechanisms of mRNA localization and localized translation near the mitochondria.

Most of mitochondrial proteins are encoded in the nucleus and need to be imported into the organelle. Import may occur while the protein is synthesized ...

Measurement and Analysis of Extracellular Acid Production to Determine Glycolytic Rate

Extracellular measurement of oxygen consumption and acid production is a simple and powerful way to monitor rates of respiration and glycolysis1. Both mitochondrial (respiration) and non-mitochondrial (other redox) reactions consume oxygen, but these reactions can be easily distinguished by chemical inhibition of mitochondrial respiration. However, while mitochondrial oxygen consumption is an unambiguous and direct measurement of respiration rate2, the same is not true for extracellular acid production and its relationship to glycolytic rate 3-6. Extracellular acid produced by cells is derived from both lactate, produced by anaerobic glycolysis, and CO2, produced in the citric acid cycle during respiration. For glycolysis, the conversion of glucose to lactate- + H+ and the export of products into the assay medium is the source of glycolytic acidification. For respiration, the export of CO2, hydration to H2CO3 and dissociation to HCO3- + H+ is the source of respiratory acidification. The proportions of glycolytic and respiratory acidification depend on the experimental conditions, including cell type and substrate(s) provided, and can range from nearly 100% glycolytic acidification to nearly 100% respiratory acidification 6. Here, we demonstrate the data collection and calculation methods needed to determine respiratory and glycolytic contributions to total extracellular acidification by whole cells in culture using C2C12 myoblast cells as a model.

Glycolysis is a defining metabolic marker in multiple biological systems. Monitoring glycolysis by measuring the extracellular flux of H+ is common, but requires correction to be quantitative and unambiguous. Here, we demonstrate how to gather and correct extracellular flux data to distinguish between respiratory and glycolytic sources of extracellular acidification.

测量外产酸和分析，以确定糖酵解率

Extracellular measurement of oxygen consumption and acid production is a simple and powerful way to monitor rates of respiration and glycolysis1. Both ...