我们要感谢以下人士没有他们的帮助不会有可能收集的松树组织：乔奈恩博士，马特布里曼，迈克尔波尔多，Ujwal Bagal，靳慧哲，阿曼达Bouffier。

第1部分：树丰收 的树被选中，砍伐后，重要的是那样认真和尽快工作 。 随着每一个不同的组织类型的收获，他们应该立即放置到自己的液氮容器，以避免任何样品材料的交叉污染。 这种RNA隔离协议的可扩展性。 <ol><li>切成约0.5-0.75米长的螺栓和电锯得分在两个或三个地方的树皮螺栓纵轴。 </li><li>使用木凿，工作取得区域的边缘，直到树皮开始从木材中分离出来，同时拉树皮的部分，继续凿，直到它完全分开干。 </li><li>次生木质部是收​​获的直边，单双面剃刀刮沿纵轴的螺栓已剥去树皮。戴乳胶手套，以尽量减少污染的组织。 </li><li>手册剥离韧皮部，这是位于树皮部分的内侧，很容易收获。 </li><li>反应木材是从四肢底面后他们用喷漆标记，表明尊重其原始方向的地面收集。 </li><li>四肢都切成1-2米的螺栓，并沿纵轴上取得的两侧，根据木材的反应两种类型的大致位置：对面的木（上顶侧肢体）和压缩木（在底部一侧肢体）。树皮中分离从上文所述的肢体，但只有树皮覆盖每种类型木材的反应是在同一时间删除。次生木质部或韧皮部，然后收集如上所述，并放置在液态氮中。 </li><li>其他样品，如针，茎尖和年轻的视锥细胞，可以通过手工或收获，利用必要的修枝剪。 </li><li>标记袋或适当的容器应放置在液氮冷冻收集的样本，并在运回实验室的干冰包装。 </li></ol>第2部分：冷冻样品铣加工<ol><li>用液态氮填充SPEX 6850冷冻厂的水库和一个研磨瓶放入影响的栏，并填写用液氮预寒意。倒入液氮和样品添加到小瓶。 </li><li>请确保没有多余的液态氮保持在研磨室和端盖前，坐在密封腔，多余的氮气是完全排出。 </li><li>插入载体和密切的研磨瓶。 </li><li>两个1分钟/循环周期率设定在14密尔木本样本。 </li><li>使用液态氮冷冻铲到一个已预冷，并含有少量的液态氮烧杯中删除的试片（木粉）。 </li><li>倒在-80℃直至所需的液态氮/样品泥浆进入储存容器和地点。 </li></ol>第3部分：RNA分离，每日1次<ol><li> RNA提取缓冲液20ML成50毫升上限猎鹰管，加400μLβ-巯基乙醇，涡旋简单地说，然后在60℃水浴热。 </li><li>每管加入4克冰冻组织。第和动摇振荡10秒手大力。 </li><li>管加入等体积的氯仿和反转轻轻混合。管放置在一个旋转的轮子，并允许最终超过年底搅拌5分钟。 </li><li>倒出到50毫升橡树岭管和12,000（17,200 XG），在一个SS34固定角度转子5分钟的转速离心机的混合物。 </li><li>水相转移到一个新的橡树岭管，并添加一个半体积的氯仿。 </li><li>涡为1分钟，继续搅拌5分钟，一个旋转的轮子上。在SS34 5分钟的固定角度转子离心机转速12,000 rpm（17,200 XG）。 </li><li>水相转移到新鲜的橡树岭管，并添加一个半体积的氯仿。涡1分钟。在SS34 10分钟的固定角度转子离心机转速12,000 rpm（17,200 XG）。 </li><li>水相转移到50 mL的高速聚丙烯管。在SS34 20分钟的固定角度转子离心机以10,000 rpm（11900 XG）。 </li><li>倒出上清液到50毫升猎鹰管，并添加一季度量10米LiCl水溶液上清。简言之涡和地方在4 ° C为过夜析出。 </li></ol>第4部分：RNA分离，第2天<ol><li>热火在65 ° C水浴开始之前SSTE缓冲区。 </li><li>倒入聚丙烯管和沉淀的RNA，用LiCl沉淀样品离心30分钟，在11000名，在一个SS34固定角度转子转速（14450 XG）4 ° C。 </li><li>离心后，倒出并丢弃液体，倒置约1分钟Kimwipes管。移液器关闭所有剩余的上清。 </li><li>在每个加热用吸管SSTE彻底混合样品800μL重悬沉淀。悬浮样品转移到2毫升Ambion公司无RNase离心管。 </li><li>热样品在65℃大力涡旋2分钟，以确保完整的再悬浮。 </li><li>加入等体积酚 - 氯仿，PH8，每个样品管。涡每个样本为30秒，然后在离心旋转，在3分钟14000转</li><li>水相转移到一个新的2ML Ambion公司无RNase离心管，加等体积的氯仿。涡每个样本为30秒，并在3分钟14,000转离心旋转。 </li><li>水相转移到锁相凝胶管先前已准备2分钟的转速在11000 microfuging。添加一个半体积的氯仿，轻轻地吸液管混合。 </li><li>离心5分钟11,000转和水相转移到新鲜2ML Ambion公司无RNase离心管。 </li></ol>第5部分：RNA沉淀和乙醇洗<ol><li>加入50微升3.0 M醋酸钠，pH值4.8，和1 mL 100％的乙醇水相。涡和沉淀在-80 ° C时为30分钟或隔夜-20 ° C。 </li><li> 14000 RPM为30分钟，在4 ° C，并小心吸上清液离心样品。请勿打扰的颗粒。 </li><li> 1毫升-20 ° C 70％（V / V）乙醇洗涤RNA的颗粒。在1分钟14000转离心和吸乙醇。 </li><li>重复步骤5.3。 </li><li> 3-5分钟替补摘帽管和空气干燥颗粒。 </li></ol>第6部分：最后再悬浮和定量<ol><li>每100μLDEPC处理水重悬沉淀。由涡旋混合，并在65 ° C孵育2分钟，然后放置在冰上管。如有必要，以确保样品的再悬浮重复。 </li><li>像如果需要的话，并为50μL1:10稀释在10MM三，pH值7.5，分光光度定量的样品池。吸光度比值为260/280和二百三十○分之二百六十○通常比分别为2.1和2.2，更大。 RNA分离单产范围从60 -120μg/ g的冷冻组织。 </li><li>运行在1％琼脂糖凝胶在TAE缓冲液1.5-2.5微克RNA​​分析的样品。更为关键的样本，分析每制造商的指示使用，安捷伦2100生物分析仪的RNA。 </li></ol>代表性的成果： RNA分离产量从不同的针叶树组织范围从60-120微克/克木本样品冰冻组织通常会产生大约70 - 80G / G冰冻组织。 260/280和二百三十分之二百六诊断率通常都超过210更大，是良好的指标，RNA样品无任何重大的酚醛树脂或碳水化合物的污染，分别。 ETBR染色的琼脂糖凝胶电泳分析的样品应显示25S，18S和5S RNA（图1）三个不同的乐队。确定的28S化学计量琼脂糖凝胶电泳18S是有些主观，凝胶，染色，样品负载运行条件等因素，都可以产生效果。在一般情况下，28S：18S的比例将显示为&gt; 1.5。然而，有些针叶样品有较低的比率。例如，安捷伦2100分析后，我们已经看到几个不同的组织类型的化学计量比接近1.2:1针叶树种样品的实例。因此，明显偏低28S：18S琼脂糖凝胶电泳化学计量并不一定表示一个质量差的样本。叶，拍摄，往往会产生更多的不同的频段目前由于叶绿体核糖体RNA锥样品。 ETBR染色的材料，不迁移以及从样品或分子质量高频段（&gt; 8-10 KB），通常表明基因组DNA污染（图2）。在5S带和减少核糖体带染色或明显18S&gt; 28S化学计量学是一个很好的指标，RNA降解发生（图3）或低于附近的低分子量涂片。在Agilent 2100电泳分析，基准线应与相应的18S和28S核糖体RNA有28S的主要峰低和相对平稳的的：18S的比例从1.2到2.0不等。安捷伦RNA的完整性号码（RIN）的值可能是一个更好的RNA质量的预测，并应至少为7或更大的RNA是用于制备芯片的目标。图4显示了一个典型的木质部与28S RNA的安捷伦2100电泳：18S比等于1.4和8.6的RIN值，而图5显示了一个穷人的木质部RNA的准备有一个非常高的基线明显下降，由28S透露：18S RA氧化钛等于0.65和3.4的RIN值。 <img src="/files/ftp_upload/1751/1751fig1.jpg" alt="图1" /> 图1：高品质的松树RNA 的琼脂糖凝胶电泳分析。 1巷2巷，NEB 1 kb的标准，显示了一个典型的总火炬松次生木质部与特色核糖体28S，18S，5S乐队和明显的28S RNA的提取：18S比值&gt; 1。 <img src="/files/ftp_upload/1751/1751fig2.jpg" alt="图2" /> 图2。松树RNA样品琼脂糖凝胶电泳分析基因组DNA的污染。 1巷，NEB 1 kb的标准，2巷，木质部显示出基因组DNA污染的RNA样品。 <img src="/files/ftp_upload/1751/1751fig3.jpg" alt="图3" /> 图3。琼脂糖凝胶电泳分析显示RNA降解和基因组DNA污染的松树样品。 1巷，NEB 1 kb的标准，Lane2，木质部呈现出基因组DNA的污染和严重的RNA降解的RNA样品。 <img src="/files/ftp_upload/1751/1751fig4.jpg" alt="图4" /> 图4。安捷伦2100木质部总RNA电泳分离出所描述的方法使用火炬松木质部。 28S：18S的比例等于1.4，RIN值等于8.6 <img src="/files/ftp_upload/1751/1751fig5.jpg" alt="图5" /> 图5。安捷伦2100呈现出严重的退化和基因组污染的根尖提示总RNA电泳。 28S：18S比值等于0.65，RIN值等于3.4

<ol>
	<li>Chang, S., Puryear, J., Cairney, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+and+efficient+method+for+extracting+RNA+from+pine+trees.">A simple and efficient method for extracting RNA from pine trees.</a> Plant Molecular Biology Reporter. 11 (2), 113-116 (1993).</li><li>Lorenz, W. W., Yu, Y. S., Sim&#369;es, M., Dean, J. F. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Processing+the+Loblolly+Pine+PtGen2+cDNA+Microarray.">Processing the Loblolly Pine PtGen2 cDNA Microarray.</a> Journal of Visualized Experiments. 25, (2009).</li></ol>

从针叶树种获得高质量的RNA可以是一个艰巨的任务，鉴于木本植物组织中发现的酚类和多糖化合物的高水平。开始与张等人开发的协议。 （1），我们已经发现，更严格的提取和清理步骤导致各种木本和非木本组织从多种针叶树种采样的质量非常高的总RNA的一致隔离。该视频已证明不仅是我们修改后的RNA分离协议，而且在实地收集和火炬松用于RNA分离前处理技术而采取的步骤。这些收获和准备步?...

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>RNA Isolation Buffer</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>2% CTAB (hexadecyltrimethylammonium bromide), 2% PVP (polyvinyl pyrrolidinone; Mw 30-40,000), 100mM Tris-HCl (pH8.0), 25mM EDTA, 2.0M NaCl, 0.5g/L Spermidine</td>
</tr>
<tr>
<td>Chloroform</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>C298-4</td>
<td>&nbsp;</td>
<tr>
<td>Phenol, Ultrapure</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15509-037</td>
<td>&nbsp;</td>
<tr>
<td>10M LiCl</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Made with DEPC-treated water</td>
</tr>
<tr>
<td>SSTE Buffer</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>1M NaCl, 0.5% SDS, 10mM Tris-HCl (pH8.0), 1mM EDTA (pH8.0)</td>
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<td>Sodium acetate (NaOAc) 3M pH4.8</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Made with DEPC-treated water</td>
</tr>
<tr>
<td>Phenol-chloroform (pH8.0)</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>120mL phenol, 160mL chloroform titrated with multiple changes of 0.5M Tris-Cl pH 8.0</td>
</tr>
<tr>
<td>Oak Ridge High Speed Teflon Tubes</td>
<td>&nbsp;</td>
<td>Thermo-Fisher</td>
<td>#05-562-16B</td>
<td>&nbsp;</td>
<tr>
<td>Polypropylene 50mL High Speed Tubes</td>
<td>&nbsp;</td>
<td>Thermo-Fisher</td>
<td>#05-562-10K</td>
<td>&nbsp;</td>
<tr>
<td>BD Falcon,sterile, capped 50mL conical disposable tubes</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>#21008-939</td>
<td>&nbsp;</td>
<tr>
<td>Phase Lock Gel Heavy, 2mL</td>
<td>&nbsp;</td>
<td>Thermo-Fisher</td>
<td>#2302830</td>
<td>&nbsp;</td>
<tr>
<td>Ambion RNase-free Microfuge Tubes, 2mL</td>
<td>&nbsp;</td>
<td>Ambion, Inc.</td>
<td>#AM12425</td>
<td>&nbsp;</td>		</tbody>
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an improved method of rna isolation from loblolly pine (p. taeda l.) and other conifer species

针叶树种，特别是那些属于松科家族，如火炬松，从组织中分离（<em&gt;火炬松</em&gt; L），含有高浓度的酚类化合物和多糖干扰RNA纯化。从这些品种的高品质的RNA分离需要严格的组织程序，收集多个有机提取步骤以孤立的芯片和其他基因组分析的质量足够的RNA组成的RNA分离协议的领域和就业。从现场收集的火炬松样本可以是具有挑战性的，但标准组织和RNA分离程序的若干修改，大大提高结果的高品质的RNA隔离。如果样品不正确收集和从外地运输，特别是在大型的收成，一般RNA降解程度增加。总RNA产量显着增加，粉碎样品在液氮冷冻轧机前RNA分离，尤其是当从木本植物组织样本。这主要是由于氧化剂的存在，如酚类化合物，多糖，都是在目前大多数针叶树种木本植物组织中提取的高层次，。如果不除去，这些污染物可以开展对领导的问题，如RNA降解，导致产量低和质量差的RNA样品。结转酚类化合物，以及多糖，还可以减少甚至完全消除cDNA合成常用的逆转录酶或其他聚合酶的活性。特别是，RNA的注定用于cDNA文库的产生双链cDNA合成，单链PCR或定量PCR的基因合成，或合成芯片的目标材料的模​​板必须是最高质量，如果研究人员期望以获得最佳的结果。常用于许多其他植物物种的RNA分离技术往往不足以从针叶样品中删除这些污染物的能力，因此不产生总RNA样品，适合于下游操作。在这个视频中，我们展示了针叶树组织进行实地收集的方法，一个四十二岁树砍伐，收获的韧皮部，次生木质部，反应木材木质部开始。我们还证明，一直随后酶操作带来了高品质的RNA的RNA分离协议。

Watch this Scientific Journal Video about An Improved Method of RNA Isolation from Loblolly Pine P. taeda L. and Other Conifer Species at JoVE.com

An Improved Method of RNA Isolation from Loblolly Pine P. taeda L. and Other Conifer Species

许多植物组织中，包括从火炬松的韧皮部及木质部（<em&gt;火炬松</em&gt; L），含有酚和干扰RNA纯化的多糖高的水平。此演示文稿讨论的收获种植现场组织和隔离的芯片和其他基因组分析的RNA质量足够技术。

的RNA分离方法的改进火炬松（ P。火炬松 L）和其他针叶树种

Tissues isolated from conifer species, particularly those belonging to the Pinaceae family, such as loblolly pine (Pinus taeda L.), contain high ...

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Research

JoVE Journal

Biology

An Improved Method of RNA Isolation from Loblolly Pine (P. taeda L.) and Other Conifer Species

13.7K 次观看.  University of Georgia (UGA). 许多植物组织中，包括从火炬松的韧皮部及木质部（火炬松 L），含有酚和干扰RNA纯化的多糖高的水平。此演示文稿讨论的收获种植现场组织和隔离的芯片和其他基因组分析的RNA质量足够技术。

视频: An Improved Method of RNA Isolation from Loblolly Pine P. taeda L. and Other Conifer Species

Processing the Loblolly Pine PtGen2 cDNA Microarray

PtGen2 is a 26,496 feature cDNA microarray containing amplified loblolly pine ESTs. The array is produced in our laboratory for use by researchers studying gene expression in pine and other conifer species. PtGen2 was developed as a result of our gene discovery efforts in loblolly pine, and is comprised of sequences identified primarily from root tissues, but also from needle and stem.1,2 PtGen2 has been tested by hybridizing different Cy-dye labeled conifer target cDNAs, using both amplified and non-amplified indirect labeling methods, and also tested with a number of hybridization and washing conditions. This video focuses on the handling and processing of slides before and after pre-hybridization, as well as after hybridization, using some modifications to procedures developed previously.3,4 Also included, in text form only, are the protocols used for the generation, labeling and clean up of target cDNA s, as well as information on software used for downstream data processing. 
PtGen2 is printed with a proprietary print buffer that contains high concentrations of salt that can be difficult to remove completely. The slides are washed first in a warm SDS solution prior to pre-hybridization. After pre-hybridization, the slides are washed vigorously in several changes of water to complete removal of remaining salts. LifterSlips&trade; are then cleaned and positioned on the slides and labeled cDNA is carefully loaded onto the microarray by way of capillary action which provides for even distribution of the sample across the slide, and reduces the chance of bubble incorporation. Hybridization of targets to the array is done at 48&deg;C in high humidity conditions. After hybridization, a series of standard washes are done at 53&deg;C and room temperature for extended times. Processing PtGen2 slides using this technique reduces salt and SDS-derived artifacts often seen when the array is processed less rigorously. Hybridizing targets derived from several different conifer RNA sources, this processing protocol yielded fewer artifacts, reduced background, and provided better consistency among different experimental groups of arrays.

The cDNA microarray PtGen2 was developed for gene expression studies in loblolly pine, P. taeda, and other conifer species. Here, we show pre- and post-hybridization handling and washing techniques that can be used with this array to yield better consistency, reduced artifacts, and lower backgrounds.

处理火炬松PtGen2差异表达基因的cDNA微阵列

PtGen2 is a 26,496 feature cDNA microarray containing amplified loblolly pine ESTs.  The array is produced in our laboratory for use by researchers ...

科研

生物学

RNA Isolation from Embryonic Zebrafish and cDNA Synthesis for Gene Expression Analysis

Many important and complex laboratory procedures require an input of high quality, intact RNA.  A degraded sample or the presence of impurities can lead to disastrous results in downstream experimental applications.  It is therefore, of utmost importance to use solid techniques with numerous safeguards and quality control checks to ensure a superior sample.  Herein, we detail a protocol to isolate total RNA from whole zebrafish embryos using a commercially available chemical denaturant and subsequent cleanup to remove traces of DNA and impurities using a commercial RNA isolation kit. As RNA is relatively unstable and easily prone to cleavage by RNAses, most protocols assay gene expression using a cDNA product that is directly synthesized from an RNA template. We detail a procedure to convert RNA into the more stable cDNA product using a commercially available kit. Throughout these procedures there are numerous quality control checks to ensure that the sample is not degraded or contaminated.  The end product of these protocols is cDNA that is suitable for microarray analysis, RT-PCR or long-term storage.    

The isolation of high quality, intact RNA is an essential step in many laboratory protocols. Here, we demonstrate RNA extraction from whole zebrafish embryos and cDNA synthesis for subsequent application in various experimental procedures including gene expression microarray analysis.

从胚胎斑马鱼和cDNA合成的基因表达分析的RNA分离

Many important and complex laboratory procedures require an input of high quality, intact RNA.  A degraded sample or the presence of impurities can lead ...

Profiling of Methyltransferases and Other S-adenosyl-L-homocysteine-binding Proteins by Capture Compound Mass Spectrometry (CCMS)

There is a variety of approaches to reduce the complexity of the proteome on the basis of functional small molecule-protein interactions such as affinity chromatography 1 or Activity Based Protein Profiling 2. Trifunctional Capture Compounds (CCs, Figure 1A) 3 are the basis for a generic approach, in which the initial equilibrium-driven interaction between a small molecule probe (the selectivity function, here S-adenosyl-L-homocysteine, SAH, Figure 1A) and target proteins is irreversibly fixed upon photo-crosslinking between an independent photo-activable reactivity function (here a phenylazide) of the CC and the surface of the target proteins. The sorting function (here biotin) serves to isolate the CC - protein conjugates from complex biological mixtures with the help of a solid phase (here streptavidin magnetic beads). Two configurations of the experiments are possible: "off-bead" 4 or the presently described "on-bead" configuration (Figure 1B). The selectivity function may be virtually any small molecule of interest (substrates, inhibitors, drug molecules).
S-Adenosyl-L-methionine (SAM, Figure 1A) is probably, second to ATP, the most widely used cofactor in nature 5, 6. It is used as the major methyl group donor in all living organisms with the chemical reaction being catalyzed by SAM-dependent methyltransferases (MTases), which methylate DNA 7, RNA 8, proteins 9, or small molecules 10. Given the crucial role of methylation reactions in diverse physiological scenarios (gene regulation, epigenetics, metabolism), the profiling of MTases can be expected to become of similar importance in functional proteomics as the profiling of kinases. Analytical tools for their profiling, however, have not been available. We recently introduced a CC with SAH as selectivity group to fill this technological gap (Figure 1A).
SAH, the product of SAM after methyl transfer, is a known general MTase product inhibitor 11. For this reason and because the natural cofactor SAM is used by further enzymes transferring other parts of the cofactor or initiating radical reactions as well as because of its chemical instability 12, SAH is an ideal selectivity function for a CC to target MTases. Here, we report the utility of the SAH-CC and CCMS by profiling MTases and other SAH-binding proteins from the strain DH5&alpha; of Escherichia coli (E. coli), one of the best-characterized prokaryotes, which has served as the preferred model organism in countless biochemical, biological, and biotechnological studies. Photo-activated crosslinking enhances yield and sensitivity of the experiment, and the specificity can be readily tested for in competition experiments using an excess of free SAH.

Profiling of Methyltransferases and Other S-adenosyl-L-homocysteine-binding Proteins by Capture Compound Mass Spectrometry CCMS

Capture Compounds are trifunctional small molecules to reduce the complexity of the proteome by functional reversible small molecule-protein interaction followed by photo-crosslinking and purification. Here we use a Capture Compound with S-adenosyl-L-homocysteine-binding as selectivity function to isolate methyltransferases from an Escherichia coli whole cell lysate and identify them by MS.

分析甲基化和其他 S腺苷 L</sub捕捉复合质谱（CCMS）>同型半胱氨酸结合蛋白

There is a variety of approaches to reduce the complexity of the proteome on the basis of functional small molecule-protein interactions such as affinity ...

Isolation and Purification of Kinesin from Drosophila Embryos

Motor proteins move cargos along microtubules, and transport them to specific sub-cellular locations. Because altered transport is suggested to underlie a variety of neurodegenerative diseases, understanding microtubule based motor transport and its regulation will likely ultimately lead to improved therapeutic approaches. Kinesin-1 is a eukaryotic motor protein which moves in an anterograde (plus-end) direction along microtubules (MTs), powered by ATP hydrolysis. Here we report a detailed purification protocol to isolate active full length kinesin from Drosophila embryos, thus allowing the combination of Drosophila genetics with single-molecule biophysical studies. Starting with approximately 50 laying cups, with approximately 1000 females per cup, we carried out overnight collections. This provided approximately 10 ml of packed embryos. The embryos were bleach dechorionated (yielding approximately 9 grams of embryos), and then homogenized. After disruption, the homogenate was clarified using a low speed spin followed by a high speed centrifugation. The clarified supernatant was treated with GTP and taxol to polymerize MTs. Kinesin was immobilized on polymerized MTs by adding the ATP analog, 5'-adenylyl imidodiphosphate at room temperature. After kinesin binding, microtubules were sedimented via high speed centrifugation through a sucrose cushion. The microtubule pellet was then re-suspended, and this process was repeated. Finally, ATP was added to release the kinesin from the MTs. High speed centrifugation then spun down the MTs, leaving the kinesin in the supernatant. This kinesin was subjected to a centrifugal filtration using a 100 KD cut off filter for further purification, aliquoted, snap frozen in liquid nitrogen, and stored at -80 &deg;C. SDS gel electrophoresis and western blotting was performed using the purified sample. The motor activity of purified samples before and after the final centrifugal filtration step was evaluated using an in vitro single molecule microtubule assay. The kinesin fractions before and after the centrifugal filtration showed processivity as previously reported in literature. Further experiments are underway to evaluate the interaction between kinesin and other transport related proteins.

Isolation and Purification of Kinesin from Drosophila Embryos

This is a protocol to isolate active full length Kinesin from Drosophila embryos for single-molecule biophysical studies. We show how to collect embryos, make the embryo lysate, and then polymerize microtubules (MTs). Kinesin is purified by immobilizing it on the MTs, spinning down the Kinesin-MT complexes, and then releasing the kinesin from the MTs via ATP addition.

kinesin的分离和纯化果蝇胚胎

Motor proteins move cargos along microtubules, and transport them to specific sub-cellular locations. Because altered transport is suggested to underlie a ...

Vampiric Isolation of Extracellular Fluid from Caenorhabditis elegans

The genetically tractable model organism C. elegans has provided insights into a myriad of biological questions, enabled by its short generation time, ease of growth and small size. This small size, though, has disallowed a number of technical approaches found in other model systems. For example, blood transfusions in mammalian systems and grafting techniques in plants enable asking questions of circulatory system composition and signaling. The circulatory system of the worm, the pseudocoelom, has until recently been impossible to assay directly. To answer questions of intercellular signaling and circulatory system composition C. elegans researchers have traditionally turned to genetic analysis, cell/tissue specific rescue, and mosaic analysis. These techniques provide a means to infer what is happening between cells, but are not universally applicable in identification and characterization of extracellular molecules. Here we present a newly developed technique to directly assay the pseudocoelomic fluid of C. elegans. The technique begins with either genetic or physical manipulation to increase the volume of extracellular fluid. Afterward the animals are subjected to a vampiric reverse microinjection technique using a microinjection rig that allows fine balance pressure control. After isolation of extracellular fluid, the collected fluid can be assayed by transfer into other animals or by molecular means. To demonstrate the effectiveness of this technique we present a detailed approach to assay a specific example of extracellular signaling molecules, long dsRNA during a systemic RNAi response. Although characterization of systemic RNAi is a proof of principle example, we see this technique as being adaptable to answer a variety of questions of circulatory system composition and signaling.

Vampiric Isolation of Extracellular Fluid from Caenorhabditis elegans

The model organism C. elegans uses pseudocoelomic fluid as a passive circulatory system. Direct assay of this fluid has not been previously possible. Here we present a novel technique to directly assay the extracellular space, and use systemic silencing signals during an RNAi response as a proof of principle example.

吸血鬼从细胞外液分离<em&gt;秀丽隐杆线虫</em

The genetically tractable model organism C. elegans has provided insights into a myriad of biological questions, enabled by its short generation time, ...

An Improved Method for Accurate and Rapid Measurement of Flight Performance in Drosophila

Drosophila has proven to be a useful model system for analysis of behavior, including flight. The initial flight tester involved dropping flies into an oil-coated graduated cylinder; landing height provided a measure of flight performance by assessing how far flies will fall before producing enough thrust to make contact with the wall of the cylinder. Here we describe an updated version of the flight tester with four major improvements. First, we added a &#34;drop tube&#34; to ensure that all flies enter the flight cylinder at a similar velocity between trials, eliminating variability between users. Second, we replaced the oil coating with removable plastic sheets coated in Tangle-Trap, an adhesive designed to capture live insects. Third, we use a longer cylinder to enable more accurate discrimination of flight ability. Fourth we use a digital camera and imaging software to automate the scoring of flight performance. These improvements allow for the rapid, quantitative assessment of flight behavior, useful for large datasets and large-scale genetic screens.

An Improved Method for Accurate and Rapid Measurement of Flight Performance in Drosophila

Here we describe a method for rapid and accurate measurement of flight performance in Drosophila, enabling high-throughput screening.

在对飞行性能的准确，快速测定方法的改进<em&gt;果蝇</em

Drosophila has proven to be a useful model system for analysis of behavior, including flight. The initial flight tester involved dropping flies into an ...

RNA Isolation from Mouse Pancreas: A Ribonuclease-rich Tissue

Isolation of high-quality RNA from ribonuclease-rich tissue such as mouse pancreas presents a challenge. As a primary function of the pancreas is to aid in digestion, mouse pancreas may contain as much a 75 mg of ribonuclease. We report modifications of standard phenol/guanidine thiocyanate lysis reagent protocols to isolate RNA from mouse pancreas. Guanidine thiocyanate is a strong protein denaturant and will effectively disrupt the activity of ribonuclease under most conditions. However, critical modifications to standard protocols are necessary to successfully isolate RNA from ribonuclease-rich tissues. Key steps include a high lysis reagent to tissue ratio, removal of undigested tissue prior to phase separation and inclusion of a ribonuclease inhibitor to the RNA solution. Using these and other modifications, we routinely isolate RNA with RNA Integrity Number (RIN) greater than 7. The isolated RNA is of suitable quality for routine gene expression analysis. Adaptation of this protocol to isolate RNA from ribonuclease rich tissues besides the pancreas should be readily achievable.

We report a procedure to isolate RNA with high integrity from the ribonuclease rich mouse pancreas.

从小鼠胰腺RNA提取：核糖核酸酶丰富的组织

Isolation of high-quality RNA from ribonuclease-rich tissue such as mouse pancreas presents a challenge.  As a primary function of the pancreas is to aid ...

Hybrid-Cut: An Improved Sectioning Method for Recalcitrant Plant Tissue Samples

Maintaining plant section integrity is essential for studying detailed anatomical structures at the cellular, tissue, or even organ level. However, some plant cells have rigid cell walls, tough fibers and crystals (calcium oxalate, silica, etc.), and high water content that often disrupt tissue integrity during plant tissue sectioning.
This study establishes a simple Hybrid-Cut tissue sectioning method. This protocol modifies a paraffin-based sectioning technique and improves the integrity of tissue sections from different plants. Plant tissues were embedded in paraffin before sectioning in a cryostat at -16 &#176;C. Sectioning under low temperature hardened the paraffin blocks, reduced tearing and scratching, and improved tissue integrity significantly. This protocol was successfully applied to calcium oxalate-rich Phalaenopsis orchid tissues as well as recalcitrant tissues such as reproductive organs and leaves of rice, maize, and wheat. In addition, the high quality of tissue sections from Hybrid-Cut could be used in combination with in situ hybridization (ISH) to provide spatial expression patterns of genes of interest. In conclusion, this protocol is particularly useful for recalcitrant plant tissue containing high crystal or silica content. Good quality tissue sections enable morphological and other biological studies.

This protocol describes a simple Hybrid-Cut tissue sectioning method that is useful for recalcitrant plant tissues. Good quality tissue sections enable anatomical studies and other biological studies including in situ hybridization (ISH).

混合式切割：用于顽拗植物组织样品的改进切法

Maintaining plant section integrity is essential for studying detailed anatomical structures at the cellular, tissue, or even organ level. However, some ...

Stomata Tape-Peel: An Improved Method for Guard Cell Sample Preparation

The study of guard cells is essential to the knowledge of the specific contributions of this cell type to the overall function of plants. However, it is often difficult to isolate and thus studying them often provides a challenge. 
This study establishes a stomatal guard cell enrichment method. The protocol utilizes Scotch tape to isolate guard cells and extract proteins from the cells. This method improves the integrity and yield of guard cell during isolation and provides a reliable sample for the study of cell signaling processes and how they correlate to stomatal movement phenotypes. In this method, the plant leaves were partitioned between two portions of tape and peeled apart to remove the abaxial side. This protocol was applied to Arabidopsis leaf tissue to isolate guard cells. The cells were treated with fluorescein diacetate (FDA) to determine viability and with abscisic acid (ABA) to assess stomata movement in response to ABA. In conclusion, this protocol proves useful for preparing enriched stomatal guard cells and isolating proteins. The quality of the cells and proteins obtained here enable physiological and other biological studies.

This protocol describes a method of preparing enriched stomatal guard cells that is useful for physiological and other biological studies.

气孔带皮: 一种改进的防护细胞样品制备方法

The study of guard cells is essential to the knowledge of the specific contributions of this cell type to the overall function of plants. However, it is ...

An Improved Time- and Labor- Efficient Protocol for Mouse Primary Hepatocyte Isolation

Primary hepatocytes are used extensively in liver in vitro research, especially in glucose metabolism studies. A base technique has been adapted based on different needs, like time, labor, cost, and primary hepatocyte usage, resulting in various primary hepatocyte isolation protocols. However, the numerous steps and time-consuming reagent preparations in primary hepatocyte isolation are major drawbacks for efficiency. After comparing different protocols for their pros and cons, the advantages of each were combined, and a rapid and efficient primary hepatocyte isolation protocol was formulated. Within only ~35 min, this protocol could yield as much, if not more, healthy primary hepatocytes as other protocols. Further, glucose metabolism experiments performed using the isolated primary hepatocytes validated the usefulness of this protocol in in vitro liver metabolism studies. We also extensively reviewed and analyzed the significance and purpose of each step in this study so that future researchers can further optimize this protocol based on needs.

Primary hepatocytes are a valuable tool to study liver response and metabolism in vitro. Utilizing commercially available reagents, an improved time- and labor-efficient protocol for mouse primary hepatocyte isolation was developed.

一种改进的小鼠原代肝细胞分离时间和劳动效率方案

Primary hepatocytes are used extensively in liver in vitro research, especially in glucose metabolism studies. A base technique has been adapted based on ...