本研究的财政支持由国家重点研究发展计划（2016YFC1200601）和国家自然科学基金（31390421）提供。

白蛉饲养和隐蔽物种识别<ol><li>在27±1℃，70±10％湿度和14小时光:10小时黑暗条件的标准条件下，将棉花棉属 （Malvaceae）（马鞭草科）（cv。Zhe-Mian 1973）保存在笼中。 </li><li>收集单个成年粉虱，并在30μL裂解缓冲液（10mM Tris，pH 8.4,50mM KCl，0.45％[wt / vol] Tween-20,0.2％[wt / vol]明胶，0.45％[vol / vol] Nonidet P 40,60g / mL蛋白酶K）。 </li><li>将匀浆在65℃孵育1小时，然后100℃孵育10分钟。 注意:如有必要，可以增加65℃的孵育时间。应严格控制100℃下的孵育时间，以充分灭活蛋白酶K并避免DNA损伤。 </li><li> 基于线粒体细胞色素氧化酶I引物，使用粉虱DNA（在1.3节获得）进行聚合酶链反应（PCR）。 COI-F:5&#39;-TTGATTTTTTGGTCATCCAGAAGT-3&#39; COI-R:5&#39;-TAATATGGCAGATTAGTGCATTGGA-3&#39; <ol><li>进行PCR反应，最终体积为25μL，含有1U Taq DNA聚合酶，2.5μL10x Buffer，0.2 mM dNTP，0.2 mM各引物和2μL粉虱DNA。 </li><li>使用以下PCR程序条件:初始变性95℃3分钟，然后35个循环，95℃45秒（变性），50℃45秒（退火）和72℃1分钟（延伸）35个循环;在72℃另外10分钟进一步延伸。 </li></ol></li><li>使用推荐的方案使用DNA凝胶提取试剂盒清洗PCR扩增产物。 </li><li>按照制造商的说明，用DNA样品测序系统对纯化的DNA样品进行顺序。 </li><li>使用基本局部比对搜索工具（BLAST）分析序列，以识别粉虱的隐蔽物种，避免其他物种的污染ES。 </li></ol>内分泌鉴定和定位<ol><li>对粉虱DNA（步骤1.3获得）进行PCR，以扩增粉虱内每个内生子的特异性基因（PCR配方在1.4节中描述，PCR程序和引物列于表1 ）。 </li><li>根据先前发布的方案20 （1％琼脂糖凝胶，Tris-乙酸盐-EDTA作为运行缓冲液，电压为5V / cm）使扩增的样品进行琼脂糖凝胶电泳，以鉴定每种细菌的特异性基因。 </li><li>恢复和序列每个条带以确定粉虱内的细菌种类（见第1.5-1.7节）。 </li><li> 在粉虱上进行荧光原位杂交（FISH），以根据先前发布的方案19来识别内共生菌的位置。 注意:如有必要，增加荧光探针的浓度。 </li></oL&gt; 透射电子显微镜（TEM） <ol><li>将含有2.5％[vol / vol]戊二醛的磷酸盐缓冲液（0.1M，pH 7.0）中的粉虱浸泡并固定4小时。 </li><li>在磷酸盐缓冲液（0.1M，pH7.0）中洗涤样品三次，每次15分钟。 </li><li>在含有1％[wt / vol] OsO 4的磷酸盐缓冲液（0.1M，pH7.0）中沉淀并固定样品2小时。 </li><li>通过一系列乙醇（30％，50％，70％，80％，90％，95％和100％），每次15分钟使样品脱水。 </li><li>将样品转移并浸入100％丙酮中20分钟。 </li><li>将样品在100％丙酮和100％Spurr树脂（1:1）的混合物中放置1小时。 </li><li>将样品转移到100％丙酮和100％Spurr树脂（1:3）的混合物中3小时。 </li><li>将样品转移并浸入100％Spurr树脂（在70℃下孵育）超过9小时。 </li><li>通过使用微型部分粉虱样品对我来说。 </li><li>通过浸渍在绝对醋酸铀酰中5-10分钟，然后浸入50％碱性柠檬酸铅另外5-10分钟，将超薄膜（在3.9节中获得）染色。 注意:第3.1-3.10节中使用的试剂的体积取决于样品。确保样品浸入试剂中。 </li><li>观察透射电子显微镜（15,000X）下的样品，以确定进一步使用的细菌的定位，形状和大小（见第4.6节）。 </li></ol>白蛉细菌解剖和纯化<ol><li>在显微镜载玻片上加入100μL1x PBS溶液。从棉花叶中挑一些3或4龄的若虫，并将其浸入PBS溶液中。 </li><li> 在立体显微镜下使用精细的昆虫针，从粉虱身上拉出细菌。 <ol><li>在若虫的一边轻轻切开一个洞，轻轻按一下一边让细菌出来。 </li></ol></li><li>在0.5-10μL移液管上装入20μL微型加载器（前端切割以满足细菌大小）。将各种细菌从PBS溶液中提取到微型加载器中。 </li><li>洗涤细菌以通过将细菌吸移到PBS溶液中并提取细菌来消除其他粉虱组织的污染。重复三次。 </li><li>立即将洗涤的细菌移至含有60μL1x PBS溶液的离心管中。 </li><li>注射器通过5μm滤膜过滤组装的细菌（由粉虱中的细菌和细菌核的大小确定）。重复多次，彻底移动混合液通过过滤膜。 注意:为了获得更好的扩增和测序结果，组装超过100个细菌。首先通过使用1×PBS溶液来过滤膜以减少细菌的损失结合膜。 </li></ol>内分泌基因组的扩增<ol><li> 使用推荐的方案，使用DNA扩增试剂盒扩增滤液（主要含有粉虱的内共生体），并进行一些修改。 <ol><li>选择从血液或细胞中扩增基因组DNA的推荐方案，并制备缓冲液D2用于随后的实验。 </li><li>直接向离心管中加入1.5μL滤液（见第4.6节），然后加入1.5μLBuffer D2，充分混匀。 </li><li>在冰上孵育10分钟，并加入1.5μL的终止溶液。 </li><li>加入15μL反应缓冲液和1μLDNA聚合酶，轻轻混匀。 </li><li>将混合物在30℃下孵育16小时，然后在65℃下孵育3分钟。 </li></ol></li><li>在扩增的滤液上直接进行PCR（如果需要，稀释扩增的样品），使用专门为bac设计的特异性引物teria来确认细菌种类（见2.1节）。 </li><li>根据先前发表的方法21 ，使用粉虱基因β-肌动蛋白和EF1引物进行PCR以确认是否存在宿主基因组的污染（PCR配方在1.4节中描述）。 </li></ol> Endosymbiont Metagenome测序<ol><li>在测序前将扩增的滤液置于分光光度计和荧光计（根据制造商的说明书），以测试样品的质量以及样品是否符合测序标准。 </li><li>根据制造商的说明书将其扩增至基因组测序仪。 </li></ol>

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Since the endosymbionts within whiteflies cannot be cultured in vitro, dissection and assembling bacteriocytes is an effective way to obtain enough genetic material of endosymbionts. Before dissection, the species of whitefly and endosymbionts involved should be explicitly confirmed. The whitefly B. tabaci is a species complex with more than 35 morphologically indistinguishable species and different cryptic species may contain different endosymbionts. Portiera is uniformly harbored as an obliga...

与节肢动物形成密切共生关系的细菌广泛存在于节肢动物中。在几乎每个发育阶段5中 ，内分泌已被证明影响宿主的方面，如营养代谢，繁殖，对环境胁迫2,3,4 等的反应。然而，支持协会的机制仍然很大程度上是未知的。研究细菌的潜在功能和作用时，基因组学是重中之重。一些基本信息， 即分类学状态，功能基因，代谢途径，分泌系统，可以从基因组序列推断出来，揭示了共生体在共生中的潜在作用。随着高通量测序的发展，大量的细菌基因组已被测序各种功能揭示6 。 内含子在半ter子中是至关重要的，如蚜虫7 ，臭虫8 ，木虱9 ，褐飞虱10和蝉11 。例如，作为专性共生体的蚜虫中的Buchnera已被证明参与必需氨基酸生物合成，以及来自蚜虫基因组12的基因。此外， Buchnera的转录调控也显示13 。在木麻黄中， 卡桑奈尔被测序，并且是发现的最小的细菌基因组14 。所有这些内含子的标志都是基于基因组序列推断的。由于这些内生子宫内膜不能在体外培养 ，因此已经采用了几种方法来分离足够的细菌uencing。在蚜虫中，通过离心和过滤提取内生子，并进行进一步的基因组和转录组学分析。在褐飞虱中，内共生连同整个昆虫基因组10进行测序。 烟粉虱是一种含有超过35种形态不可区分物种（隐蔽物种）的物种，其中两种入侵物种已经入侵到世界各地，对农业生产造成了巨大的危害15 。值得注意的是， 烟粉虱物种内的内生梭菌在害虫16的发育中已显示出重要性。到目前为止，在粉虱中已经鉴定出8个内含子，其中包括专性共生体， Candidatus Portiera aleyrodidarum和七个次生共生体Hamiltonella，立克次体 ， Arsenophonus ， Cardinium ，Wolbachia， Fritschea和Hemipteriphilus定义为17,18 。 与以前描述的半扁桃不同，粉虱烟粉虱是一只极小的昆虫，长度只有1毫米。大多数内生子体被限制在细菌19 （含有共生体的特殊细胞，其进一步在烟粉虱中形成细菌）。此外，这些内生子宫内膜不能在体外培养 。从烟粉虱获得内生菌的唯一方法是将细菌分解出来。然而，解剖有困难。首先，脆弱的细菌总是与粉虱的其他组织连接，这是很难分离的。其次，粉虱的微小尺寸限制了足够细菌的分离。第三，内生菌群聚集在细菌中，使获得单一种细菌非常复杂。

<table border="0" cellpadding="0" cellspacing="0" width="775">
	<tbody>
		<tr height="53">
			<td height="53" style="height:53px;width:243px;">Taq DNA polymerase</td>
			<td style="width:107px;">Takara</td>
			<td style="width:129px;">R001A</td>
			<td style="width:296px;">including rTaq, 10&#215;Buffer and dNTP</td>
		</tr>
		<tr height="53">
			<td height="53" style="height:53px;width:243px;">Gel DNA extraction kit</td>
			<td style="width:107px;">Qiagen</td>
			<td style="width:129px;">28704</td>
			<td></td>
		</tr>
		<tr height="53">
			<td height="53" style="height:53px;width:243px;">DNA sample sequencing system</td>
			<td style="width:107px;">ABI</td>
			<td style="width:129px;">ABI-3730XL</td>
			<td></td>
		</tr>
		<tr height="53">
			<td height="53" style="height:53px;width:243px;">Microtome</td>
			<td style="width:107px;">Leica</td>
			<td style="width:129px;">EM UC7</td>
			<td></td>
		</tr>
		<tr height="53">
			<td height="53" style="height:53px;width:243px;">Transmission electron microscopy</td>
			<td style="width:107px;">Hitachi</td>
			<td style="width:129px;">H-7650 TEM</td>
			<td></td>
		</tr>
		<tr height="53">
			<td height="53" style="height:53px;width:243px;">Stereo microscope</td>
			<td style="width:107px;">Zeiss</td>
			<td style="width:129px;">Stemi 2000-C</td>
			<td></td>
		</tr>
		<tr height="53">
			<td height="53" style="height:53px;width:243px;">20 &#956;L microloader</td>
			<td style="width:107px;">Eppendorf</td>
			<td style="width:129px;">F2771951</td>
			<td></td>
		</tr>
		<tr height="53">
			<td height="53" style="height:53px;width:243px;">Filter holder</td>
			<td style="width:107px;">Millipore</td>
			<td style="width:129px;">SX0001300</td>
			<td></td>
		</tr>
		<tr height="53">
			<td height="53" style="height:53px;width:243px;">Filter membrane filter</td>
			<td style="width:107px;">Millipore</td>
			<td style="width:129px;">SMWP001300</td>
			<td>5.0 &#956;m SMWP</td>
		</tr>
		<tr height="53">
			<td height="53" style="height:53px;width:243px;">REPLI-g UltraFast Mini Kit</td>
			<td style="width:107px;">Qiagen</td>
			<td style="width:129px;">150033</td>
			<td>DNA amlification kit</td>
		</tr>
		<tr height="53">
			<td height="53" style="height:53px;width:243px;">NanoDrop</td>
			<td style="width:107px;">Thermo Scientific</td>
			<td style="width:129px;">NanoDrop 2000</td>
			<td></td>
		</tr>
		<tr height="53">
			<td height="53" style="height:53px;">Qubit Fluorometer</td>
			<td style="width:107px;">Thermo Fisher Scientific</td>
			<td style="width:129px;">Q33216</td>
			<td></td>
		</tr>
		<tr height="53">
			<td height="53" style="height:53px;width:243px;">Genome Sequencer</td>
			<td style="width:107px;">Illumina</td>
			<td style="width:129px;">Hiseq 2000</td>
			<td></td>
		</tr>
	</tbody>
</table>

methods for the extraction of endosymbionts from the whitefly bemisia tabaci

细菌共生体与宿主形成亲密关系，在大多数情况下赋予宿主优势。基因组信息对于研究其宿主细菌共生体的功能和进化至关重要。由于大多数共生体不能在体外培养 ，所以分离足够量的细菌用于基因组测序的方法是非常重要的。在粉虱Bemisia tabaci中 ，已经确定了许多内生子，并且通过多种方法预测害虫的发育和繁殖是重要的。然而，支持协会的机制仍然很大程度上是未知的。障碍部分来自于粉虱中的内分泌物，主要是抑制细菌，难以与宿主细胞分离。在这里我们报告一个主要通过解剖的粉虱B.烟粉虱鉴定，提取和纯化内分泌物的分步骤方案开和过滤。通过该方法制备的内含子样品虽然仍然是不同内分泌物种的混合物，但适用于随后的基因组测序和分析烟粉虱内共生菌的可能作用。这种方法也可用于从其他昆虫中分离内分泌物。

以烟草烟草复合体的中东亚洲1号（MEAM1）为例进行说明。用于饲养粉虱的棉花和几个发育阶段的粉虱显示在图1中，包括棉花植物，成年粉虱和粉虱的第1， 第 2和第 4龄的若虫（3龄的若虫似乎与第4龄若虫相似） ）。显然，第4龄若虫比第1龄和第2龄的若虫大（ 图1 ）。 MEAM1中Portiera和Hamiltonella的 FISH?...

Watch this Scientific Journal Video about Methods for the Extraction of Endosymbionts from the Whitefly Bemisia tabaci at JoVE.com

Methods for the Extraction of Endosymbionts from the Whitefly Bemisia tabaci

在这里，我们提出了通过解剖和过滤从粉虱烟粉虱中分离内分泌物的方案。扩增后，DNA样品适用于后续测序和研究内共生与粉虱之间的共生关系。

从白蛉提取内分泌物的方法<em&gt; Bemisia tabaci</em

Bacterial symbionts form an intimate relationship with their hosts and confer advantages to the hosts in most cases. Genomic information is critical to ...

methods-for-extraction-endosymbionts-from-whitefly-bemisia

Research

JoVE Journal

Biology

Methods for the Extraction of Endosymbionts from the Whitefly Bemisia tabaci

9.8K 次观看.  Zhejiang University. 在这里，我们提出了通过解剖和过滤从粉虱烟粉虱中分离内分泌物的方案。扩增后，DNA样品适用于后续测序和研究内共生与粉虱之间的共生关系。 

视频: Methods for the Extraction of Endosymbionts from the Whitefly Bemisia tabaci

Methods for Patch Clamp Capacitance Recordings from the Calyx

We demonstrate the basic techniques for presynaptic patch clamp recording at the calyx of Held, a mammalian central nervous system nerve terminal. Electrical recordings from the presynaptic terminal allow the measurement of action potentials, calcium channel currents, vesicle fusion (exocytosis) and subsequent membrane uptake (endocytosis). The fusion of vesicles containing neurotransmitter causes the vesicle membrane to be added to the cell membrane of the calyx. This increase in the amount of cell membrane is measured as an increase in capacitance. The subsequent reduction in capacitance indicates endocytosis, the process of membrane uptake or removal from the calyx membrane. Endocytosis, is necessary to maintain the structure of the calyx and it is also necessary to form vesicles that will be filled with neurotransmitter for future exocytosis events. Capacitance recordings at the calyx of Held have made it possible to directly and rapidly measure vesicular release and subsequent endocytosis in a mammalian CNS nerve terminal. In addition, the corresponding postsynaptic activity can be simultaneously measured by using paired recordings. Thus a complete picture of the presynaptic and postsynaptic electrical activity at a central nervous system synapse is achievable using this preparation. Here, the methods for slice preparation, morphological features for identification of calyces of Held, basic patch clamping techniques, and examples of capacitance recordings to measure exocytosis and endocytosis are presented.

We demonstrate the basic techniques for presynaptic patch clamp recording at the calyx of Held, a mammalian central nervous system nerve terminal.

膜片钳从花萼电容录音方法

We demonstrate the basic techniques for presynaptic patch clamp recording at the calyx of Held, a mammalian central nervous system nerve terminal. ...

科研

生物学

Micro-dissection of Rat Brain for RNA or Protein Extraction from Specific Brain Region

Micro-dissection of rat brain into various regions is extremely important for the study of different neurodegenerative diseases. This video demonstrates micro-dissection of four major brain regions include olfactory bulb, frontal cortex, striatum and hippocampus in fresh rat brain tissue. Useful tips for quick removal of respective regions to avoid RNA and protein degradation of the tissue are given.

Micro-dissection of rat brain into various regions is extremely important for the study of different neurodegenerative diseases. This video demonstrates micro-dissection of four major brain regions include olfactory bulb, frontal cortex, striatum and hippocampus in fresh rat brain tissue. Useful tips for quick removal of respective regions to avoid RNA and protein degradation of the tissue are given.

从特定的大脑区域的RNA或蛋白质提取大鼠脑微解剖

Micro-dissection of rat brain into various regions is extremely important for the study of different neurodegenerative diseases. This video demonstrates ...

RNA Extraction from Neuroprecursor Cells Using the Bio-Rad Total RNA Kit

使用Bio - Rad公司的总RNA试剂盒从Neuroprecursor细胞的RNA提取

Electrophysiological Methods for Recording Synaptic Potentials from the NMJ of Drosophila Larvae

In this video, we describe the electrophysiological methods for recording synaptic transmission at the neuromuscular junction (NMJ) of Drosophila larva. The larval neuromuscular system is a model synapse for the study of synaptic physiology and neurotransmission, and is a valuable research tool that has defined genetics and is accessible to experimental manipulation. Larvae can be dissected to expose the body wall musculature, central nervous system, and peripheral nerves. The muscles of Drosophila and their innervation pattern are well characterized and muscles are easy to access for intracellular recording. Individual muscles can be identified by their location and orientation within the 8 abdominal segments, each with 30 muscles arranged in a pattern that is repeated in segments A2 - A7. Dissected drosophila larvae are thin and individual muscles and bundles of motor neuron axons can be visualized by transillumination1. Transgenic constructs can be used to label target cells for visual identification or for manipulating gene products in specific tissues. In larvae, excitatory junction potentials (EJP&#8217;s) are generated in response to vesicular release of glutamate from the motoneurons at the synapse. In dissected larvae, the EJP can be recorded in the muscle with an intracellular electrode. Action potentials can be artificially evoked in motor neurons that have been cut posterior to the ventral ganglion, drawn into a glass pipette by gentle suction and stimulated with an electrode. These motor neurons have distinct firing thresholds when stimulated, and when they fire simultaneously, they generate a response in the muscle. Signals transmitted across the NMJ synapse can be recorded in the muscles that the motor neurons innervate. The EJP&#8217;s and minature excitatory junction potentials (mEJP&#8217;s) are seen as changes in membrane potential. Electrophysiological responses are recorded at room temperature in modified minimal hemolymph-like solution2 (HL3) that contains 5 mM Mg2+ and 1.5 mM Ca2+. Changes in the amplitude of evoked EJP&#8217;s can indicate differences in synaptic function and structure. Digitized recordings are analyzed for EJP amplitude, mEJP frequency and amplitude, and quantal content.

Here we describe electrophysiological methods for measuring synaptic transmission at the neuromuscular junction of Drosophila larva. Evoked release is initiated artificially by stimulating the motor neuron axons, and transmission through the NMJ can be measured by the postsynaptic response evoked in the muscle.

电生理方法记录从果蝇幼虫NMJ突触潜力

In this video, we describe the electrophysiological methods for recording synaptic transmission at the neuromuscular junction (NMJ) of Drosophila larva. ...

Methods for the Study of the Zebrafish Maxillary Barbel

Barbels are skin sensory appendages found in fishes, reptiles and amphibians. The zebrafish, Danio rerio, develops two pairs of barbels- a short nasal pair and a longer maxillary pair. Barbel tissue contains cells of ectodermal, mesodermal and neural crest origin, including skin cells, glands, taste buds, melanocytes, circulatory vessels and sensory nerves. Unlike most adult tissue, the maxillary barbel is optically clear, allowing us to visualize the development and maintenance of these tissue types throughout the life cycle. 
This video shows early development of the maxillary barbel (beginning approximately one month post-fertilization) and demonstrates a surgical protocol to induce regeneration in the adult appendage (&gt;3 months post-fertilization). Briefly, the left maxillary barbel of an anesthetized fish is elevated with sterile forceps just distal to the caudal edge of the maxilla. A fine, sterile spring scissors is positioned against the forceps to cut the barbel shaft at this level, establishing an anatomical landmark for the amputation plane. Regenerative growth can be measured with respect to this plane, and in comparison to the contralateral barbel. Barbel tissue regenerates rapidly, reaching maximal regrowth within 2 weeks of injury.
Techniques for analyzing the regenerated barbel include dissecting and embedding matched pairs of barbels (regenerate and control) in the wells of a standard DNA electrophoresis gel. Embedded specimens are conveniently photographed under a stereomicroscope for gross morphology and morphometry, and can be stored for weeks prior to downstream applications such as paraffin histology, cryosectioning, and/or whole mount immunohistochemistry. These methods establish the maxillary barbel as a novel in vivo tissue system for studying the regenerative capacity of multiple cell types within the genetic context of zebrafish.

The zebrafish maxillary barbel is an integumentary sense organ containing ectodermal, mesodermal and neural crest derivatives. Importantly, the adult barbel can regenerate after proximal amputation. This video introduces maxillary barbel development and demonstrates a surgical protocol to induce regeneration, followed by collection, embedding and downstream imaging of barbel specimens.

上颌Barbel斑马鱼的研究方法

Barbels are skin sensory appendages found in fishes, reptiles and amphibians.  The zebrafish, Danio rerio, develops two pairs of barbels- a short nasal ...

Extraction of the EPP Component from the Surface EMG

A surface electromyogram (EMG), especially when recorded near the neuromuscular junction, is expected to contain the endplate potential (EPP) component which can be extracted with an appropriate signal filter. Two factors are important: the EMG must be recorded in monopolar fashion, and the recording must be done so the low frequency signal corresponding the EPP is not eliminated. This report explains how to extract the EPP component from the EMG of the masseter muscle in a human subject. The surface EMG is recorded from eight sites using traditional disc electrodes aligned along over the muscle, with equal inter-electrode distance from the zygomatic arch to the angle of mandible in response to quick gum clenching. A reference electrode is placed on the tip of the nose. The EPP component is extracted from the raw EMGs by applying a high-cut digital filter (2nd dimension Butterworth filter) with a range of 10-35 Hz. When the filter is set to 10 Hz, the extracted EPP wave deflects either negative or positive depending on the recording site. The difference in the polarity reflects the sink-source relation of the end plate current, with the site showing the most negative deflection corresponding to the neuromuscular junction. In the case of the masseter muscle, the neuromuscular junction is estimated to be located in the inferior portion close to the angle of mandible. The EPP component exhibits an interesting oscillation when the cut-off frequency of the high-cut digital filter is set to 30 Hz. The EPP oscillation indicates that muscle contraction is adjusted in an intermittent manner. Abnormal tremors accompanying various sorts of diseases may be substantially due to this EPP oscillation, which becomes slower and is difficult to cease.

The endplate potential (EPP) component can be extracted from the surface EMG using a digital filter. The extracted EPP shows oscillation with a frequency of about 30 Hz.

从表面肌电信号的提取资源增值计划的组件

A surface electromyogram (EMG), especially when recorded near the neuromuscular junction, is expected to contain the endplate potential (EPP) component ...

Budding Yeast Protein Extraction and Purification for the Study of Function, Interactions, and Post-translational Modifications

Homogenization by bead beating is a fast and efficient way to release DNA, RNA, proteins, and metabolites from budding yeast cells, which are notoriously hard to disrupt. Here we describe the use of a bead mill homogenizer for the extraction of proteins into buffers optimized to maintain the functions, interactions and post-translational modifications of proteins. Logarithmically growing cells expressing the protein of interest are grown in a liquid growth media of choice. The growth media may be supplemented with reagents to induce protein expression from inducible promoters (e.g. galactose), synchronize cell cycle stage (e.g. nocodazole), or inhibit proteasome function (e.g. MG132). Cells are then pelleted and resuspended in a suitable buffer containing protease and/or phosphatase inhibitors and are either processed immediately or frozen in liquid nitrogen for later use. Homogenization is accomplished by six cycles of 20 sec&#160;bead-beating (5.5 m/sec), each followed by one minute incubation on ice. The resulting homogenate is cleared by centrifugation and small particulates can be removed by filtration. The resulting cleared whole cell extract (WCE) is precipitated using 20% TCA for direct analysis of total proteins by SDS-PAGE followed by Western blotting. Extracts are also suitable for affinity purification of specific proteins, the detection of post-translational modifications, or the analysis of co-purifying proteins. As is the case for most protein purification protocols, some enzymes and proteins may require unique conditions or buffer compositions for their purification and others may be unstable or insoluble under the conditions stated. In the latter case, the protocol presented may provide a useful starting point to empirically determine the best bead-beating strategy for protein extraction and purification. We show the extraction and purification of an epitope-tagged SUMO E3 ligase, Siz1, a cell cycle regulated protein that becomes both sumoylated and phosphorylated, as well as a SUMO-targeted ubiquitin ligase subunit, Slx5.

The preparation of high quality yeast cell extracts is a necessary first step in the analysis of individual proteins or entire proteomes. Here we describe a fast, efficient, and reliable homogenization protocol for budding yeast cells that has been optimized to preserve protein functions, interactions, and post-translational modifications.

芽殖酵母蛋白质的提取和纯化功能研究，交互和翻译后修饰

Homogenization by bead beating is a fast and efficient way to release DNA, RNA, proteins, and metabolites from budding yeast cells, which are notoriously ...

The Infiltration-centrifugation Technique for Extraction of Apoplastic Fluid from Plant Leaves Using Phaseolus vulgaris as an Example

The apoplast is a distinct extracellular compartment in plant tissues that lies outside the plasma membrane and includes the cell wall. The apoplastic compartment of plant leaves is the site of several important biological processes, including cell wall formation, cellular nutrient and water uptake and export, plant-endophyte interactions and defence responses to pathogens. The infiltration-centrifugation method is well established as a robust technique for the analysis of the soluble apoplast composition of various plant species. The fluid obtained by this method is commonly known as apoplast washing fluid (AWF). The following protocol describes an optimized vacuum infiltration and centrifugation method for AWF extraction from Phaseolus vulgaris (French bean) cv. Tendergreen leaves. The limitations of this method and the optimization of the protocol for other plant species are discussed. Recovered AWF can be used in a wide range of downstream experiments that seek to characterize the composition of the apoplast and how it varies in response to plant species and genotype, plant development and environmental conditions, or to determine how microorganisms grow in apoplast fluid and respond to changes in its composition.

The Infiltration-centrifugation Technique for Extraction of Apoplastic Fluid from Plant Leaves Using Phaseolus vulgaris as an Example

This protocol details the optimized extraction of apoplast washing fluid from plant leaves, using French bean plants (Phaseolus vulgaris) as a model example.

渗透离心技术质外体中提取植物液使用叶<em&gt;菜豆</em&gt;为例

The apoplast is a distinct extracellular compartment in plant tissues that lies outside the plasma membrane and includes the cell wall. The apoplastic ...

Filtration Isolation of Nucleic Acids:  A Simple and Rapid DNA Extraction Method

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad to extract cellular DNA from whole blood in less than 2 min. The blood specimen is treated with detergent, mixed briefly and applied by pipet to the separation membrane. The lysate wicks into the blotting pad due to capillary action, capturing the genomic DNA on the surface of the separation membrane. The extracted DNA is retained on the membrane during a simple wash step wherein PCR inhibitors are wicked into the absorbent blotting pad. The membrane containing the entrapped DNA is then added to the PCR reaction without further purification. This simple method does not require laboratory equipment and can be easily implemented with inexpensive laboratory supplies. Here we describe a protocol for highly sensitive detection and quantitation of HIV-1 proviral DNA from 100 &#181;l whole blood as a model for early infant diagnosis of HIV that could readily be adapted to other genetic targets.

We describe here a simple and rapid paper-based DNA extraction method of HIV proviral DNA from whole blood detected by quantitative PCR. This protocol can be extended for use in detecting other genetic markers or using alternative amplification methods.

核酸的分离过滤:一种简单快速的DNA提取方法

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad ...

Methods for the Isolation, Culture, and Functional Characterization of Sinoatrial Node Myocytes from Adult Mice

Sinoatrial node myocytes (SAMs) act as the natural pacemakers of the heart, initiating each heart beat by generating spontaneous action potentials (APs). These pacemaker APs reflect the coordinated activity of numerous membrane currents and intracellular calcium cycling. However the precise mechanisms that drive spontaneous pacemaker activity in SAMs remain elusive. Acutely isolated SAMs are an essential preparation for experiments to dissect the molecular basis of cardiac pacemaking. However, the indistinct anatomy, complex microdissection, and finicky enzymatic digestion conditions have prevented widespread use of acutely isolated SAMs. In addition, methods were not available until recently to permit longer-term culture of SAMs for protein expression studies. Here we provide a step-by-step protocol and video demonstration for the isolation of SAMs from adult mice. A method is also demonstrated for maintaining adult mouse SAMs in vitro and for expression of exogenous proteins via adenoviral infection. Acutely isolated and cultured SAMs prepared via these methods are suitable for a variety of electrophysiological and imaging studies.

Methods are demonstrated for the isolation of sinoatrial node myocytes (SAMs) from adult mice for patch clamp electrophysiology or imaging studies. Isolated cells can be used directly or can be maintained in culture to permit expression of proteins of interest, such as genetically encoded reporters.

从成年小鼠的分离，培养和窦房结肌细胞的功能分析方法

Sinoatrial node myocytes (SAMs) act as the natural pacemakers of the heart, initiating each heart beat by generating spontaneous action potentials (APs). ...