我们感谢R.迈克尔Sramkoski博士的帮助，流式细胞仪，主任Purnima Jai​​swal博士和博士翟武以前的输入和伊利亚·Aylyarov插图的帮助。支持的由NIH资助R01GM089872的和流式细胞术和显微成像技术的核心设施的案例综合癌症中心（P30 CA43703）。

1。单倍体细胞生长<ol><li>成长S.从的S288C背景（BY4741）的酵母细胞在标准条件下10。 </li><li>变换单倍体两个显眼的荧光标记物10的任一表达。给出的标记物是在细胞质中表达的可溶性的GFP（pEG220，URA3/YIp，11用BglII线性化）和一个单一的核糖体蛋白，RPL25 mCherry（pAJ1661，URA3/CEN，从A约翰斯顿）融合。我们预计，相当于两色的纯化方法可以利用其他明亮的荧光蛋白质合成的单倍体亲本细胞。可替代地，细胞壁可以标记用荧光的外源凝集素。 </li><li>前一天受精卵净化，接种5毫升的文化，不同的细胞类型和成长振荡培养过夜，在23°C，达到A600不超过1.0。成长GFP的的转化（ATY4442）完整的合成培养基中，包括2％葡萄糖（CSM-葡萄糖）ND RPL25-mCherry的转化（ATY4552）的合成选择性培养基中缺乏尿嘧啶10。保持平行每种类型的细胞的培养物，在室温下振荡。 </li></ol> 2。开展跨<ol><li>于新鲜培养基中，调整每个样品的光密度（A600）为1.0。 [1光密度单位〜3×10 7个细胞/ ml] </li><li>混合等量的两种文化，涡简要的3倍，其次是一个较长的漩涡，持续10秒。 </li><li>准备CSM-葡萄糖板。该板用于常规生长的那些是相同的，并已被干燥45分钟后，浇注和凝固琼脂10。 </li><li>分发多达二十40毫升的混合物1厘米从CSM-葡萄糖板的表面的边缘的液滴。当液滴首先被施加到表面的启动的定时器，以测量经过的时间。一旦液体被吸收（大约30分钟），覆盖板，让他们不受干扰地在州m温度。 </li></ol> 3。收获的准备工作<ol><li>收获1.75-2.5小时后，在23°C的样品落盘使用150毫升冷CSM-葡萄糖，保持板置于冰上，通过反复洗涤个人干的液滴。反复冲洗液滴面积的10-20倍，具有相同的介质，以最大限度地恢复。收集在15ml Falcon管中，在冰上。 </li><li>探头的Fisher FB120（120Watt，120Volt，频率20KHZ）超声波仪，冰超声处理10倍的样本，在60％的幅度。 </li><li>在相差显微镜验证，很少或根本没有聚集。 15-50％的细胞被预期在这一点上已经形成的受精卵。 </li><li>离开该管的细胞不受干扰在冰上10分钟作为一个进一步的预防措施（允许聚集定居）在垂直位置。 </li><li>恢复最顶端的90％的体积和筛选，通过尼龙网（BD的猎鹰聚丙烯管滤网盖（编号352235））。在一个典型的实验中，10 7个细胞从一个板回收4毫升CSM-葡萄糖稀释成在这一点上。 </li></ol> 4。流式细胞仪<ol><li>排序细胞与一个100微米的小费使用索尼iCyt反射模型流式细胞仪（高度自动化的并行排序）HAPS1的。 GFP激发，使用蓝色的氩离子电池，250毫瓦的激光波长为488 nm。 mCherry激励，使用100毫瓦的激光波长561 nm。虽然我们没有与其他机器的经验，我们预计，Becton Dickinson公司的FACSAria，，贝克顿迪克森的流入或Beckman Coulter公司MoFlo XDP时，将被罚款配备正确的激光器。 </li><li>底座排序参数上的前进与后退的散点图，以门上的活细胞。建立一个排放情节为615 + / - 30 nm的带通滤波器（mCherry）与525 + / - 25 nm的带通滤波器（GFP），以划分为四个象限的事件，“红+绿的”，“红+绿+”，“红 - 绿色“和”红-绿+“（ 图S2）。使用无荧光的细胞作为一个标准的F或确定的“红 - 绿 - ”象限。 </li><li>排序细胞的鞘21.5 psi的压力，液滴43,400滴/秒的频率，和一个样本20.5 psi的压力。 </li><li> 初始排序（“恢复模式”）：收集样品放入冷冻Eppendorf管中含有250毫升的冷CSM-葡萄糖。沉积物10秒在顶端在冷藏的离心速度之间（Tomy MRX-150，15,000 rpm下）和吸液的流动溶液（流式细胞仪鞘液（PBS，用1mM EDTA）BioSure产品目录号：1027）。重悬在500毫升冷CSM-葡萄糖。 </li><li> 第二类（“纯模式”）：初始排序进行排序，使用“纯度模式”选项。沉积物的纯化的细胞，并重新悬浮在500毫升1.0毫升CSM-葡萄糖的粒料。在23℃振摇15分钟，在24孔板中以允许代谢恢复。 </li></ol> 5。 DeltaVision的显微镜<ol><li>如下：准备琼脂糖凝胶垫带悬挂的1.5％琼脂糖（超纯琼脂糖，Invitrogen公司，产品编号15510-07）CSM-葡萄糖的水烧开，旋涡震荡，然后申请的琼脂糖溶液0.3毫升样品水平标准玻片的表面，覆盖在一次与第二个幻灯片（不施加压力）。 </li><li>冷却10分钟后，轻轻地取下上滑动，水平滑动，照顾，留下一个光滑的表面，而不是扰乱琼脂糖层。 （当已被移除的上部滑动时，细胞的样品应在1-2分钟之内施加当不除去上滑动的情况下保持在室温下在潮湿的腔室时，焊盘可以被存储为使用前一天）。 </li><li>沉积物样品和存款1ml等分的粒料在琼脂糖焊盘（不接触该表面的情况下）。立即用正方形的1号盖玻片覆盖样品。修剪掉多余的琼脂糖与单刃的剃胡刀。用凡士林密封准备从注射器分配。 </li><li>图片使用DeltaVision的（应用实现了n）的100倍油浸物镜没有的分级（奥林巴斯UPlanApo 100x/1.40;∞/ 0.17/FN26.5），。 </li><li>反卷积Z-的堆栈使用softWoRx 5.0.0的的程序（ <a href="http://www.api.com/softworx.asp" target="_blank">http://www.api.com/softworx.asp</a> ）和处理微创。通常收集在0.2-0.4微米的间隔连续的Z平面。 </li></ol> 6。代表性的成果 在转化的单倍体细胞：在单倍体的荧光水平的荧光强度水平示出在图S1。在十字架上，落射荧光检查显示，基本上所有的GFP转化为绿色和mCherry转化，虽然往往是微弱的，是红色的。 ，以确保所有细胞用于交叉确实荧光，我们使用了亚群与亮的荧光信号。 产量：初始排序后，红色+绿色+样品（ 图S2）典型地包括8-20％的输入的细胞数和过多数量的单倍体细胞，也许是由于持久性的细胞-细胞粘连。由于高富集与单一排序，还没有实现的部分纯化的样品应该是探头超声处理两次在60％的振幅，然后通过重新排序。 （恢复各种通常持续1-2小时之间，而第二类需要15分钟。）的细胞受到的第二类，红+绿+样本包括输入单元格的23-52％。 相镜检：考试相差显微镜显示，21岁以上的准备（从86％到91％不等），平均约90％的纯度的最终受精卵准备。的这些污染物的绿色或红色的单倍体，或双单倍体，在彼此接触的， 图3列出了％的受精卵具有无芽，内侧芽集团（Med），的侧芽（纬度）或顶芽（定期），成对的相同的颜色的单倍体（“2Same”）或不同的颜色（的“2Hap”）的数目，以及个别单倍体（厦门）。 1.75小时后，收获细胞，当超过50％是unbudded和其他亚类（内侧，外侧，或顶芽）同样表示。 2.5小时后，有大约相同数目的受精卵在四类（unbudded，内侧，外侧或顶芽）。 显微观察（图4）： 受精卵的结构出现在纯化过程中保持不变。根据十字架的持续时间，人口有不同的的比例unbudded和发芽受精卵，如上所述。因为两者的绿色和红色的单倍体荧光信号强度表现出相当大的变化，一些受精卵主要是红色，而另一些则主要是绿色，因为是由开始比较观察g为在图4中的合成图像的单色图像。还请注意，绿色荧光蛋白（分子量26kDa）进入细胞核，并且可以发现即使当细胞核没有融合在细胞核中。液泡站出来为黑色非荧光球。 最终产品的纯化，在不同的发展阶段会产生不同的人口。合子标记为A没有经历融合。 B型受精卵unbudded。 C型有一个小芽。 D型有较大的萌芽状态，E型受精卵进行细胞分裂。还请注意，许多早期的受精卵仍然表现出了非荧光内侧分割线（*），并在液泡中的“继承结构”（I）12，经常可以看到从受精卵到芽延长。在某些情况下，绿信号已到伙伴重新分配到更大的程度，加上红色信号。这反映了传输延迟，那是典型的多聚核糖体（Tartakoff，在准备中）。 6个的受精卵，这是足够的许多免疫印迹，使用化学发光的检测程序。使用苯酚 - 氯仿隔离协议我们从这个数目的细胞中提取的RNA约1.5毫克。 由于纯化的受精卵已被冷却，进一步的分析之前，我们建议在23℃下在生长培养基中培养。 <img alt="图1" src="/files/ftp_upload/4197/4197fig1.jpg" /> 图1。受精卵的形成。合子单倍体与二倍体之间的中间年表 。此图显示了单倍体经典激活（shmooing），他们的融合，而形成的最初的萌芽。芽可以内侧（M），横向（L）或终端（T）。 <imgALT =“图1”SRC =“/ files/ftp_upload/4197/4197fig2.jpg”/&gt; 图2。合子的纯化方案的原理图。孵育后，在完全培养基中与葡萄糖琼脂平板的表面上，通过洗涤回收的细胞混合物中，然后进行第二个排序在“纯度模式随后在”恢复模式“的初始排序 。 “黄色表示mCherry和GFP信号在受精卵中可见。 <img alt="图3" src="/files/ftp_upload/4197/4197fig3.jpg" /> 图3。合子纯组分交配混合物品种收获后1.75或2.5小时恢复在比较早的与比较成熟的受精卵丰富的分数。八个独立的实验中，所指定的不同的颜色，进行每个时间点。图表显示的相对数量受精卵公顷VE空间芽形成不同的模式。鉴于在培养的短暂持续时间（小于2.5小时，在室温下）观察到的所有芽初始合子出芽事件。 <img alt="图4" src="/files/ftp_upload/4197/4197fig4.jpg" /> 图4。 DeltaVision的检查丰富的分数。一个典型的领域进行了检查，Deltavision显微镜。该图显示红色和绿色的或单一的颜色。加上红色对应的多聚核糖体的标签（Rpl25p-mCherry）和胞浆GFP绿色。注意：高浓缩的受精卵，并在指定的亚细胞功能的空泡（V），花蕾（B），核（N），空泡“的继承结构”（我），分区（*）出现分离的父母域处于比较初级的受精卵。他们的变量的整体颜色反映的单倍体融合的相对强度。在案件中，两亲本域保留不同的颜色，平衡的标记（细胞质GFP，mCherry标签的多聚核糖体）是不完整的。时间的推移正在进行中的研究表明，可溶性GFP重新分配之前，多聚核糖体。 <a href="http://www.jove.com/files/ftp_upload/4197/4197fig4large.jpg" target="_blank">点击此处查看大图</a> 。 补充传奇<img alt="图S1" src="/files/ftp_upload/4197/4197figS1.jpg" /> 图S1。荧光的单倍体细胞。左：表达mCherry标签核糖体的细胞进行检查，以检测一个绿色的信号。矩形窗口被设置为避免阴性细胞。中图：胞内GFP的表达进行了分析，积极的显着明亮的重大人口。最右边的表达mCherry标记核糖体进行了分析。的矩形（在最左边的相同）包括最亮的细胞，并允许所有要避免的底片。指定“R2”表示所指示的矩形内回收的细胞的百分比。 <img alt="图S2" src="/files/ftp_upload/4197/4197figS2.jpg" /> 图S2。细胞分布在第一和第二分拣。这些两色图表明在右上象限（R 5）绿+红+细胞的比例。在第二个排序，这个比例上升到52％。水平轴：绿色信号。垂直轴：红色信号。

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没有利益冲突的声明。

获得纯化的受精卵应包括转录组学，蛋白质组学和脂类组学，以及调查机制，促进生化研究的受精卵进行重新进入细胞周期，细胞壁重塑，萌芽和继承的特点，另外，受精卵可以开始生成与承载特性突变株在父母一方或双方。此外，纯化的受精卵，为单倍体或二倍体细胞，也被冻结在含15％甘油的培养基和购买解冻监察方面经常出芽。 使用其他适合微观目的的荧光标记物（ <e...

<table border="1"><tbody><tr><td> 试剂/设备 </td><td> 源 </td><td> 目录编号 </td></tr><tr><td> pEG220，URA3/YIp，编码细胞质GFP 11） </td><td> E.格罗特</td><td></td></tr><tr><td> pAJ1661，URA3/CEN，RPL25编码融合mCherry </td><td> A.约翰斯顿</td><td></td></tr><tr><td>费舍尔FB120超声波仪</td><td> Fisher Scientific则</td><td></td></tr><tr><td>聚丙烯管滤网盖</td><td> BD猎鹰</td><td>参考352235 </td></tr><tr><td>冷藏的离心</td><td> TOMY </td><td> MRX-150 </td></tr><tr><td>流式细胞仪鞘液</td><td> Biosure </td><td> 1027 </td></tr><tr><td> iCyt反射模型流式细胞仪</td><td>索尼</td><td></td></tr><tr><td> DeltaVision的反卷积显微镜</td><td>磷肥recision </td><td></td></tr><tr><td>直立式落射荧光显微镜</td><td>徕卡</td><td></td></tr><tr><td>超纯琼脂糖</td><td> Invitrogen公司</td><td> 15510-07 </td></tr><tr><td> CSM葡萄糖配方 注：如果在琼脂平板上，简单地包括2％琼脂（Difco） </td><td> 10 </td><td></td></tr></tbody></table>

flow cytometry-based purification of s. cerevisiae zygotes

合子是必不可少的中间体单倍体和二倍体国家之间在许多生物的生命周期，包括酵母（ 图1）1。S. 酵母合子从不同的交配型的单倍体细胞（MATa的，MATalpha）的融合的结果，并产生相应的稳定的二倍体，依次产生多达20二倍体后代作为结果惊人地不对称的有丝分裂2。 Zygote的形成是由一个复杂的事件序列策划：在这个过程中，可溶性交配因子结合到同源受体，触发受体介导的信号转导通路的有利于在细胞 - 细胞融合的细胞周期和达到高潮中断。受精卵可考虑的祖代细胞或干细胞的功能的模型。 虽然已经了解到的形成受精卵，受精卵已用于研究细胞分子的问题，一般显“接力，几乎所有的研究都使用的交配混合物，其中的受精卵混合与人口占多数的单倍体细胞3-8。合子形成的生物化学的研究的许多方面和合子继续寿命，因此，仍然得不到澄清。 报告的纯化酵母受精卵的描述协议根据其沉降性，但是，这种沉淀为基础的程序没有取得近90％的纯度在我们手中。此外，它具有的缺点，即细胞暴露于高渗山梨糖醇。因此，我们已经开发出一种通用的纯化方法。为了这个目的，双单倍体细胞表达红色或绿色荧光蛋白共同孵育，以允许合子形成，在不同时间收获的，和所得到的受精卵进行纯化，使用流式细胞仪基于排序协议。这种技术提供了一个方便的可视化评估的纯度和成熟。平均纯度的馏分，是约90％。根据收获的时间，受精卵的成熟程度不一，可以恢复。纯化的样品提供了一个方便的“组学”研究的出发点，为恢复初始后代，这个祖细胞，并进行系统的调查。

Watch this Scientific Journal Video about Flow Cytometry-based Purification of S. cerevisiae Zygotes at JoVE.com

Flow Cytometry-based Purification of S. cerevisiae Zygotes

为了净化受精卵<em&gt; S.酵母</em&gt;，相反交配型的单倍体细胞的设计，以表达红色或绿色荧光蛋白，共同孵育，以允许合子形成，并分级的使用基于流式细胞术的协议。高度富集的部分，随后的“组学”的研究，恢复初始的后代，受精卵形态和系统的调查。

流式细胞仪分离纯化<em&gt; S.酵母</em&gt;合子

Zygotes are essential intermediates between haploid and diploid states in the life cycle of many organisms, including yeast (Figure 1) 1. S. cerevisiae ...

flow-cytometry-based-purification-of-s-cerevisiae-zygotes

Research

JoVE Journal

Biology

Flow Cytometry-based Purification of S. cerevisiae Zygotes

12.7K 次观看.  Case Western Reserve University School of Medicine. 为了净化受精卵 S.酵母，相反交配型的单倍体细胞的设计，以表达红色或绿色荧光蛋白，共同孵育，以允许合子形成，并分级的使用基于流式细胞术的协议。高度富集的部分，随后的“组学”的研究，恢复初始的后代，受精卵形态和系统的调查。

视频: Flow Cytometry-based Purification of S. cerevisiae Zygotes

Assay for Adhesion and Agar Invasion in S. cerevisiae

Yeasts are found in natural biofilms, where many microorganisms colonize surfaces. In artificial environments, such as surfaces of man-made objects, biofilms can reduce industrial productivity, destroy structures, and threaten human life. 1-3 On the other hand, harnessing the power of biofilms can help clean the environment and generate sustainable energy. 4-8
The ability of S. cerevisiae to colonize surfaces and participate in complex biofilms was mostly ignored until the rediscovery of the differentiation programs triggered by various signaling pathways and environmental cues in this organism. 9, 10 The continuing interest in using S. cerevisiae as a model organism to understand the interaction and convergence of signaling pathways, such as the Ras-PKA, Kss1 MAPK, and Hog1 osmolarity pathways, quickly placed S. cerevisiae in the junction of biofilm biology and signal transduction research. 11-20 To this end, differentiation of yeast cells into long, adhesive, pseudohyphal filaments became a convenient readout for the activation of signal transduction pathways upon various environmental changes. However, filamentation is a complex collection of phenotypes, which makes assaying for it as if it were a simple phenotype misleading. In the past decade, several assays were successfully adopted from bacterial biofilm studies to yeast research, such as MAT formation assays to measure colony spread on soft agar and crystal violet staining to quantitatively measure cell-surface adherence. 12, 21 However, there has been some confusion in assays developed to qualitatively assess the adhesive and invasive phenotypes of yeast in agar.
Here, we present a simple and reliable method for assessing the adhesive and invasive quality of yeast strains with easy-to-understand steps to isolate the adhesion assessment from invasion assessment. Our method, adopted from previous studies, 10, 16 involves growing cells in liquid media and plating on differential nutrient conditions for growth of large spots, which we then wash with water to assess adhesion and rub cells completely off the agar surface to assess invasion into the agar. We eliminate the need for streaking cells onto agar, which affects the invasion of cells into the agar. In general, we observed that haploid strains that invade agar are always adhesive, yet not all adhesive strains can invade agar medium. Our approach can be used in conjunction with other assays to carefully dissect the differentiation steps and requirements of yeast signal transduction, differentiation, quorum sensing, and biofilm formation.

We describe a qualitative assay for yeast adhesion and agar invasion as a measure of invasive and pseudohyphal differentiation. This simple assay can be used to assess the invasive phenotype of various mutants as well as the effects environmental cues and signaling pathways on yeast differentiation.

化验粘附和酿酒酵母琼脂入侵

Yeasts are found in natural biofilms, where many microorganisms colonize surfaces. In artificial environments, such as surfaces of man-made objects, ...

科研

生物学

A high-throughput method to globally study the organelle morphology in S. cerevisiae

High-throughput methods to examine protein localization or organelle morphology is an effective tool for studying protein interactions and can help achieve an comprehensive understanding of molecular pathways. In Saccharomyces cerevisiae, with the development of the non-essential gene deletion array, we can globally study the morphology of different organelles like the endoplasmic reticulum (ER) and the mitochondria using GFP (or variant)-markers in different gene backgrounds. However, incorporating GFP markers in each single mutant individually is a labor-intensive process. Here, we describe a procedure that is routinely used in our laboratory. By using a robotic system to handle high-density yeast arrays and drug selection techniques, we can significantly shorten the time required and improve reproducibility. In brief, we cross a GFP-tagged mitochondrial marker (Apc1-GFP) to a high-density array of 4,672 nonessential gene deletion mutants by robotic replica pinning.  Through diploid selection, sporulation, germination and dual marker selection, we recover both alleles. As a result, each haploid single mutant contains Apc1-GFP incorporated at its genomic locus. Now, we can study the morphology of mitochondria in all non-essential mutant background. Using this high-throughput approach, we can conveniently study and delineate the pathways and genes involved in the inheritance and the formation of organelles in a genome-wide setting.

GFP-fusion proteins are widely used to visualize organelles by confocal microscopy. However, screening for mutations that affect the morphology of organelles generally requires individual mutagenesis and is time consuming. Here, we demonstrate a method to simultaneously incorporate organelle-GFP markers in almost 5,000 non-essential genes in yeast.

一种高通量的方法，在全球范围内研究在酿酒酵母中的细胞器形态

High-throughput methods to examine protein localization or organelle morphology is an effective tool for studying protein interactions and can help ...

Identification of protein complexes with quantitative proteomics in S. cerevisiae

Lipids are the building blocks of cellular membranes that function as barriers and in compartmentalization of cellular processes, and recently, as important intracellular signalling molecules. However, unlike proteins, lipids are small hydrophobic molecules that traffic primarily by poorly described nonvesicular routes, which are hypothesized to occur at membrane contact sites (MCSs). MCSs are regions where the endoplasmic reticulum (ER) makes direct physical contact with a partnering organelle, e.g., plasma membrane (PM). The ER portion of ER-PM MCSs is enriched in lipid-synthesizing enzymes, suggesting that lipid synthesis is directed to these sites and implying that MCSs are important for lipid traffic. Yeast is an ideal model to study ER-PM MCSs because of their abundance, with over 1000 contacts per cell, and their conserved nature in all eukaryotes. Uncovering the proteins that constitute MCSs is critical to understanding how lipids traffic is accomplished in cells, and how they act as signaling molecules. We have found that an ER called Scs2p localize to ER-PM MCSs and is important for their formation. We are focused on uncovering the molecular partners of Scs2p. Identification of protein complexes traditionally relies on first resolving purified protein samples by gel electrophoresis, followed by in-gel digestion of protein bands and analysis of peptides by mass spectrometry. This often limits the study to a small subset of proteins. Also, protein complexes are exposed to denaturing or non-physiological conditions during the procedure. To circumvent these problems, we have implemented a large-scale quantitative proteomics technique to extract unbiased and quantified data. We use stable isotope labeling with amino acids in cell culture (SILAC) to incorporate staple isotope nuclei in proteins in an untagged control strain. Equal volumes of tagged culture and untagged, SILAC-labeled culture are mixed together and lysed by grinding in liquid nitrogen. We then carry out an affinity purification procedure to pull down protein complexes. Finally, we precipitate the protein sample, which is ready for analysis by high-performance liquid chromatography/ tandem mass spectrometry. Most importantly, proteins in the control strain are labeled by the heavy isotope and will produce a mass/ charge shift that can be quantified against the unlabeled proteins in the bait strain. Therefore, contaminants, or unspecific binding can be easily eliminated. By using this approach, we have identified several novel proteins that localize to ER-PM MCSs. Here we present a detailed description of our approach. 

Here we describe a new quantitative proteomics technique for identifying protein complexes in Saccharomyces cerevisiae. In this study, we have used the SILAC method together with affinity purification followed by tandem mass spectrometry to identify with high specificity the binding partners of an ER protein, Scs2p.

鉴定与定量蛋白质组学在酿酒酵母中的蛋白质复合物

Lipids are the building blocks of cellular membranes that function as barriers and in compartmentalization of cellular processes, and recently, as ...

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents (Methanol, Ethanol or 2-Propanol) and acetic acid are used for fixation, staining and destaining of proteins in a gel after SDS-PAGE. To speed up the procedure, heating the staining solution in the microwave oven for a short time is frequently used. This usually results in evaporation of toxic or hazardous Methanol, Ethanol or 2-Propanol and a strong smell of acetic acid in the lab which should be avoided due to safety considerations. In a protocol originally published in two patent applications by E.M. Wondrak (US2001046709 (A1), US6319720 (B1)), an alternative composition of the staining solution is described in which no organic solvent or acid is used. The CBB is dissolved in bidistilled water (60-80mg of CBB G-250 per liter) and 35 mM HCl is added as the only other compound in the staining solution. The CBB staning of the gel is done after SDS-PAGE and thorough washing of the gel in bidistilled water. By heating the gel during the washing and staining steps, the process can be finished faster and no toxic or hazardous compunds are evaporating. The staining of proteins occurs already within 1 minute after heating the gel in staining solution and is fully developed after 15-30 min with a slightly blue background that is destained completely by prolonged washing of the stained gel in bidistilled water, without affecting the stained protein bands.

A short protocol for protein staining with Coomassie Brilliant Blue (CBB) G-250 in polyacrylamide gels is described without using organic solvents or acetic acid as in the classical staining procedures with CBB.

无有机溶剂和醋酸，蛋白质与考马斯亮蓝G - 250凝胶染色

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

Flow Cytometry Purification of Mouse Meiotic Cells

The heterogeneous nature of cell types in the testis and the absence of meiotic cell culture models have been significant hurdles to the study of the unique differentiation programs that are manifest during meiosis. Two principal methods have been developed to purify, to varying degrees, various meiotic fractions from both adult and immature animals: elutriation or Staput (sedimentation) using BSA and/or percoll gradients. Both of these methods rely on cell size and density to separate meiotic cells1-5. Overall, except for few cell populations6, these protocols fail to yield sufficient purity of the numerous meiotic cell populations that are necessary for detailed molecular analyses. Moreover, with such methods usually one type of meiotic cells can be purified at a given time, which adds an extra level of complexity regarding the reproducibility and homogeneity when comparing meiotic cell samples.
Here, we describe a refined method that allows one to easily visualize, identify, and purify meiotic cells, from germ cells to round spermatids, using FACS combined with Hoechst 33342 staining7,8. This method provides an overall snapshot of the entire meiotic process and allows one to highly purify viable cells from most stage of meiosis. These purified cells can then be analyzed in detail for molecular changes that accompany progression through meiosis, for example changes in gene expression9,10and the dynamics of nucleosome occupancy at hotspots of meiotic recombination11.

An efficient method to obtain highly purified viable meiotic fractions from mouse testis is described, which combines a refined cell dissociation protocol with fluorescent activated cell sorting (FACS). This method takes advantage of differences in the DNA content and nuclear density of discrete meiotic fractions.

流动小鼠减数分裂细胞的流式细胞仪净化

The heterogeneous nature of cell types in the testis and the absence of meiotic cell culture models have been significant hurdles to the study of the ...

Studying Age-dependent Genomic Instability using the S. cerevisiae Chronological Lifespan Model

Studies using the Saccharomyces cerevisiae aging model have uncovered life span regulatory pathways that are partially conserved in higher eukaryotes1-2. The simplicity and power of the yeast aging model can also be explored to study DNA damage and genome maintenance as well as their contributions to diseases during aging. Here, we describe a system to study age-dependent DNA mutations, including base substitutions, frame-shift mutations, gross chromosomal rearrangements, and homologous/homeologous recombination, as well as nuclear DNA repair activity by combining the yeast chronological life span with simple DNA damage and mutation assays. The methods described here should facilitate the identification of genes/pathways that regulate genomic instability and the mechanisms that underlie age-dependent DNA mutations and cancer in mammals.

Studying Age-dependent Genomic Instability using the S. cerevisiae Chronological Lifespan Model

Here we describe a set of DNA mutation assays that can be combined with the yeast chronological life span model to study the genes/pathways that regulate or contribute to genomic DNA instability during aging.

研究年龄相关的基因组不稳定性使用 S。酵母年代的寿命模型

Studies using the Saccharomyces cerevisiae aging model have uncovered life span regulatory pathways that are partially conserved in higher eukaryotes1-2. ...

Expression and Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein in Saccharomyces cerevisiae

The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel, that when mutated, can give rise to cystic fibrosis in humans.There is therefore considerable interest in this protein, but efforts to study its structure and activity have been hampered by the difficulty of expressing and purifying sufficient amounts of the protein1-3. Like many 'difficult' eukaryotic membrane proteins, expression in a fast-growing organism is desirable, but challenging, and in the yeast S. cerevisiae, so far low amounts were obtained and rapid degradation of the recombinant protein was observed 4-9. Proteins involved in the processing of recombinant CFTR in yeast have been described6-9 .In this report we describe a methodology for expression of CFTR in yeast and its purification in significant amounts. The protocol describes how the earlier proteolysis problems can be overcome and how expression levels of CFTR can be greatly improved by modifying the cell growth conditions and by controlling the induction conditions, in particular the time period prior to cell harvesting. The reagants associated with this protocol (murine CFTR-expressing yeast cells or yeast plasmids) will be distributed via the US Cystic Fibrosis Foundation, which has sponsored the research. An article describing the design and synthesis of the CFTR construct employed in this report will be published separately (Urbatsch, I.; Thibodeau, P. et al., unpublished). In this article we will explain our method beginning with the transformation of the yeast cells with the CFTR construct - containing yeast plasmid (Fig. 1). The construct has a green fluorescent protein (GFP) sequence fused to CFTR at its C-terminus and follows the system developed by Drew et al. (2008)10. The GFP allows the expression and purification of CFTR to be followed relatively easily. The JoVE visualized protocol finishes after the preparation of microsomes from the yeast cells, although we include some suggestions for purification of the protein from the microsomes. Readers may wish to add their own modifications to the microsome purification procedure, dependent on the final experiments to be carried out with the protein and the local equipment available to them. The yeast-expressed CFTR protein can be partially purified using metal ion affinity chromatography, using an intrinsic polyhistidine purification tag. Subsequent size-exclusion chromatography yields a protein that appears to be &gt;90% pure, as judged by SDS-PAGE and Coomassie-staining of the gel.

Expression and Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein in Saccharomyces cerevisiae

Attempts to express the cystic fibrosis transmembrane conductance regulator (CFTR) in Saccharomyces cerevisiae have, until now, yielded relatively low amounts of protein. This protocol and the associated reagents distributed via the Cystic Fibrosis Foundation should allow the preparation of milligram amounts of this 'difficult' eukaryotic membrane protein.

囊性纤维化跨膜传导调节蛋白表达和纯化酿酒酵母

The  cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride  channel, that when mutated, can give rise to cystic fibrosis in  ...

Optimized Staining and Proliferation Modeling Methods for Cell Division Monitoring using Cell Tracking Dyes

Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of different cell types in vitro and in vivo.1-5 Although there are literally thousands of publications using such dyes, some of the most commonly encountered cell tracking applications include monitoring of:
<ol>
	<li>stem and progenitor cell quiescence, proliferation and/or differentiation6-8</li>
	<li>antigen-driven membrane transfer9 and/or precursor cell proliferation3,4,10-18 and</li>
	<li>immune regulatory and effector cell function1,18-21.</li>
</ol>
Commercially available cell tracking dyes vary widely in their chemistries and fluorescence properties but the great majority fall into one of two classes based on their mechanism of cell labeling. "Membrane dyes", typified by PKH26, are highly lipophilic dyes that partition stably but non-covalently into cell membranes1,2,11. "Protein dyes", typified by CFSE, are amino-reactive dyes that form stable covalent bonds with cell proteins4,16,18. Each class has its own advantages and limitations. The key to their successful use, particularly in multicolor studies where multiple dyes are used to track different cell types, is therefore to understand the critical issues enabling optimal use of each class2-4,16,18,24.
The protocols included here highlight three common causes of poor or variable results when using cell-tracking dyes. These are:
<ol>
	<li>Failure to achieve bright, uniform, reproducible labeling. This is a necessary starting point for any cell tracking study but requires attention to different variables when using membrane dyes than when using protein dyes or equilibrium binding reagents such as antibodies.</li>
	<li>Suboptimal fluorochrome combinations and/or failure to include critical compensation controls. Tracking dye fluorescence is typically 102 - 103 times brighter than antibody fluorescence. It is therefore essential to verify that the presence of tracking dye does not compromise the ability to detect other probes being used.</li>
	<li>Failure to obtain a good fit with peak modeling software. Such software allows quantitative comparison of proliferative responses across different populations or stimuli based on precursor frequency or other metrics. Obtaining a good fit, however, requires exclusion of dead/dying cells that can distort dye dilution profiles and matching of the assumptions underlying the model with characteristics of the observed dye dilution profile.</li>
</ol>
Examples given here illustrate how these variables can affect results when using membrane and/or protein dyes to monitor cell proliferation.

Successful use of cell tracking dyes to monitor immune cell function and proliferation involves several critical steps. We describe methods for: 1) obtaining bright, uniform, reproducible label-ing with membrane dyes; 2) selecting fluorochromes and data acquisition conditions; and 3) choosing a model to quantify cell proliferation based on dye dilution.

优化细胞分裂监测细胞示踪染料的染色和扩散建模方法

Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of ...

High Throughput Microinjections of Sea Urchin Zygotes

Microinjection into cells and embryos is a common technique that is used to study a wide range of biological processes. In this method a small amount of treatment solution is loaded into a microinjection needle that is used to physically inject individual immobilized cells or embryos. Despite the need for initial training to perform this procedure for high-throughput delivery, microinjection offers maximum efficiency and reproducible delivery of a wide variety of treatment solutions (including complex mixtures of samples) into cells, eggs or embryos. Applications to microinjections include delivery of DNA constructs, mRNAs, recombinant proteins, gain of function, and loss of function reagents. Fluorescent or colorimetric dye is added to the injected solution to enable instant visualization of efficient delivery as well as a tool for reliable normalization of the amount of the delivered solution. The described method enables microinjection of 100-400 sea urchin zygotes within 10-15 min.

Microinjection is a common technique used to deliver DNA constructs, mRNAs, morpholino antisense oligonucleotides or other treatments into eggs, embryos, and cells of various species.

海胆受精卵的高通量显微注射

Microinjection into cells and embryos is a common technique that is used to study a wide range of biological processes. In this method a small amount of ...

Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein Expressed in Saccharomyces cerevisiae

Defects in the cystic fibrosis transmembrane conductance regulator (CFTR) protein cause cystic fibrosis (CF), an autosomal recessive disease that currently limits the average life expectancy of sufferers to &#60;40 years of age. The development of novel drug molecules to restore the activity of CFTR is an important goal in the treatment CF, and the isolation of functionally active CFTR is a useful step towards achieving this goal.
We describe two methods for the purification of CFTR from a eukaryotic heterologous expression system, S. cerevisiae. Like prokaryotic systems, S. cerevisiae can be rapidly grown in the lab at low cost, but can also traffic and posttranslationally modify large membrane proteins. The selection of detergents for solubilization and purification is a critical step in the purification of any membrane protein. Having screened for the solubility of CFTR in several detergents, we have chosen two contrasting detergents for use in the purification that allow the final CFTR preparation to be tailored to the subsequently planned experiments.
In this method, we provide comparison of the purification of CFTR in dodecyl-&#946;-D-maltoside (DDM) and 1-tetradecanoyl-sn-glycero-3-phospho-(1&#39;-rac-glycerol) (LPG-14). Protein purified in DDM by this method shows ATPase activity in functional assays. Protein purified in LPG-14 shows high purity and yield, can be employed to study post-translational modifications, and can be used for structural methods such as small-angle X-ray scattering and electron microscopy. However it displays significantly lower ATPase activity.

Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein Expressed in Saccharomyces cerevisiae

Heterologous expression and purification of the cystic fibrosis transmembrane conductance regulator (CFTR) are significant challenges and limiting factors in the development of drug therapies for cystic fibrosis. This protocol describes two methods for the isolation of milligram quantities of CFTR suitable for functional and structural studies.&#160;