我们感谢所​​有成员的Kerscher实验室的鼎力支持。我们感谢：马克Hochstrasser酵母菌株MHY3765的。这项工作是由NSF资助1051970（OK）和霍华德·休斯医学研究所授予本科旅游和梦露学者项目资助（ES）的支持。

纯化6xHis-tagged蛋白表达在芽殖酵母细胞在天然条件下 1。酵母细胞的生长和诱导蛋白表达 （修改自2） 可选：使用对数生长的酵母培养物表达感兴趣的蛋白质，而不是下面描述的半乳糖诱导的培养。 <ol><li>转化细胞的半乳糖+酵母菌株的质粒编码半乳糖诱导的6xHis标签的蛋白给出。例如，试剂列表。 </li><li>合适的选择性培养基（ 如 SD-尿嘧啶）在5毫升含2％蔗糖，接种转化体。孵育在30°CO / N，旋转。 </li><li>使稀释的O / N培养物在600nm处的光密度测量0.3（OD 600 = 0.3），33毫升的选择性培养基中，用2％蔗糖。增长在30°C，晃动（Δ150转）。 </li><li>当培养已达到OD 600 = 0.8-1.5，通过加入17毫升的3倍的YEP 6％的半乳糖（配方在表1中），以使最终浓度为1x YEP用2％的半乳糖诱导表达所需蛋白。总培养体积是现在50毫升。孵育，振摇，在30℃下5-6小时。 注意：培养体积可以变化。在步骤1.3，稀释成所需的最终体积的三分之二的。在步骤1.4中，加入三分之一的最终体积的3倍YEP / 6％的半乳糖。 </li><li>测量OD 600诱导培养物的离心Δ150-200 OD 600单位的细胞在4℃下以5,000 rpm 5分钟</li><li>重悬细胞沉淀用1 ml冰冷的1X PBS 1X蛋白酶抑制剂和转移到2毫升的螺丝帽筒。 </li><li>离心细胞在4℃下以15,000 rpm的1分钟倒出上清。 </li><li>在液氮中速冻细胞沉淀，随后立即细胞裂解或储存于-80℃下，直到进一步使用。 </li></ol><ol><li>前一步骤中的冷冻细胞沉淀，加入200μl的酸洗过的玻璃珠，加入500μl冰冷的裂解缓冲液（配方在表1中，或使用细胞裂解缓冲液中给出）。 </li><li>简言之吸取向上和向下。它不要求完全重悬细胞沉淀。冰在任何时候都保持管。 注：枪头年底前可能会被切掉上下吹打。 </li><li>在4℃，地方管（S）与细胞成珠磨机，平衡，锁，并按照生产商的指示机器运行。 </li><li>运行含珠打浆机管（次），持续20秒，在5.5米/秒，然后放置在泥泞的冰，持续1分钟。总重复6X。 </li><li>清除所提取的蛋白质通过在15,000 rpm下离心15分钟，在4℃下可选：离心通过一个SpinX过滤去除小颗粒。 </li><li>准备样品的全细胞Extract（WCE）的感兴趣的蛋白质的Western印迹，以确认存在： <ol><li>添加WCE对应的2 OD 600单位的单元格（ 例如，5微升清零的提取物，如果200 OD 600单位的细胞收获），于800微升20％三氯乙酸（TCA）。旋涡混合。 </li><li>在4℃下以15,000 rpm离心2.5分钟倒出上清，但小心保留沉淀。 </li><li>添加800微升2％三氯乙酸颗粒与涡管，然后离心2.5分钟，以15,000 rpm于4℃。倒出上清，但小心保留沉淀。 </li><li>添加100微升TCA样品缓冲液（配方见表1），涡旋解散颗粒。注：如果样品缓冲液变成酸性（除了沉淀后变成黄色），加等分Δ10微升Tris碱[1M]直到样品再次是蓝色。 </li><li>孵育2-5分钟，在100℃加热块。 </li><li>涡AG泉充分溶解，如果仍然存在残余颗粒。用这种方法制备的颗粒饲料是出了名的难以充分溶解。这可能需要Δ10分钟涡旋颗粒完全溶解。 </li><li>商店样品在-80°C，直到进一步的使用。 </li></ol></li><li>急速冷冻，直到进一步利用剩余清除WCE在-80°C的液态氮和存储等分。 </li></ol> 3。批量纯化蛋白酵母细胞匀浆注：此纯化方法进行了优化，CO 2 +金属亲和树脂纯化6xHis-tagged蛋白。 <ol><li> 树脂平衡 <ol><li>清除WCEΔ20-40 OD 600个单位的细胞制备的样品，加50-100μl的亲和树脂离心管。提取从细胞中不表达所需蛋白或一种非特异性的树脂，如直链淀粉，蛋白质可被用作控制S非特异性结合。 </li><li>用1ml的洗涤缓冲液洗涤树脂5倍：翻转顶部比底部，直到树脂再悬浮，然后在每分钟5000转旋转1分钟，在4℃下吸出上清液。 注意：如果在同一天进行提取和纯化，树脂的平衡可以在提取前进行。 </li></ol></li><li> 亲和纯化的蛋白结合 <ol><li>加入100-200微升的澄清的裂解液（Δ20-40 OD 600单位），以50〜100微升的洗涤过 ​​的有孔玻璃珠，使总体积为1毫升用裂解缓冲液。 </li><li>章动或摆动，在4℃孵育2-5小时。 </li><li>自旋为1分钟，在5,000 rpm在4℃下</li><li>如果需要的话，除未结合的蛋白，用于随后的分析的样品的剩余的上清液。三氯乙酸沉淀详细在WCE（步骤2.6）以上。 </li><li>洗树脂与结合蛋白5倍用1ml的洗涤缓冲液在5,000 rpm，1分钟，然后通过旋4℃下在洗涤过程中保持样品冷。 </li></ol></li><li> 洗脱结合蛋白 <ol><li>树脂加入150μl的洗脱缓冲液，章动，在4℃下5分钟，自旋为1分钟，在5,000 rpm在4℃下保存一个新的管中的上层清液。可选：重复2倍和游泳池洗脱。 </li><li>准备洗脱样品的Western blot分析：将25μl洗脱的蛋白质，加入25μl的2倍的锂十二烷基硫酸钠（LDS）样品缓冲液中与2μl的β-巯基乙醇（BME），并在100℃的加热块中孵育2分钟。 注意：也可以使用标准的2X Laemmli样品缓冲液。 </li><li>速冻多余洗脱蛋白在液氮。 可选：带材的剩余树脂的蛋白质与等体积的2×LDS样品缓冲液中于65℃下进行5分钟，取出LDS洗脱的蛋白质，以一个新的管，然后加入2微升的BME的洗脱蛋白，而不是到树脂。此命令是为了防止拆除任何离子或抗体共轭到珠。 </li><li>样品储存在-80°C，直到进一步利用。 </li></ol></li><li> 可视化蛋白质印迹和探针与相应的抗体 。 <ol><li>装入10-20μl的各样品给出的SDS-PAGE凝胶中的蛋白质入水口和3-10微升（Δ1-3 OD 600单位）。常规用于此协议包括4-12％的Bis-Tris和8％Tris-甘氨酸凝胶。 </li><li>在200伏50分钟运行凝胶。 </li><li>半干转移蛋白质从凝胶转移到PVDF膜上，在19伏特，约20-30分钟（在表1中的配方）。 </li><li> 1小时RT或O / N挡膜在4％milk/1x的Tris缓冲生理盐水吐温（TBST）在4°C（配方见表1）。 </li><li>在4℃下孵育膜与初级抗体的表位标记的感兴趣的蛋白在4％milk/1x，TBST为1-3小时，在室温下或O / N </li><li>各5分钟，用1×TBST洗膜3倍。 </li><li>适当的孵育膜次级辣根过氧化物酶（HRP）标记的抗体为1-3小时，在RT。 </li><li>为各15分钟，在一个大的体积为1x TBST洗膜3倍。 </li><li> ，ECL底和包裹在保鲜膜覆盖膜。 </li><li>公开膜膜，并开发可视化蛋白质。 </li></ol></li></ol>

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该协议的重点编制完整的，原生的，和翻译后修饰的蛋白质从芽殖酵母细胞为下游应用。然后再尝试该协议，以确定是否可以很容易地在变性条件下12制备的蛋白提取物中检测到感兴趣的蛋白质，这是至关重要的。如果不可用多克隆抗体，它可能是有利的，使感兴趣的蛋白质的表位标记的融合蛋白的Western印迹上可检测到。在本协议中，我们标记SIZ1与医管局的6xHis-V5，或myc表位的标?...

酵母遗传学的令人敬畏的力量是有口皆碑的，但是从芽殖酵母， 酿酒酵母，天然蛋白的制备和分析往往是充满了问题。后者是由于相当大的机械强度和弹性的酵母细胞壁1。已经描述了不同的手段的酶 ​​，化学，机械，和基于压力的破坏的酵母细胞获得全细胞蛋白提取物2-6。这些技术有很大的不同，在它们的功效产生代表细胞，天然蛋白质提取物，可用于随后的分析或纯化步骤。例如，可以去除酵母细胞壁溶解酶（ 如酶解酶）和由此产生的spheroblasts会受到剪切，洗涤剂，或渗透裂解，释放蛋白。这种方法已经成功地采用了许多亚细胞分馏的起点，但它需要漫长的孵化与一些蛋白7的稳定性是不兼容的。 专有的化学提取的酵母细胞的蛋白质的的酵母裂解试剂（如清洁剂）是市售的，但在蛋白提取这些试剂的功效在随后的蛋白质的生化特性和其效果并不总是很清楚8。高压均质机，通常被称为法国印刷机，有效突破首先对他们进行高压，然后挤出来通过一个小口压力细胞的酵母细胞。该技术生产出高品质的提取物，但设备是非常昂贵的，可能不适合小批量的细胞或多个样品9。因此，机械破碎酵母细胞在珠磨机往往是原生酵母蛋白制剂10给出的方法。这项技术涉及的酵母细胞壁的机械破碎，用酸娃流下的玻璃珠，它可以与各种振动筛，旋涡仪或珠磨机进行。值得注意的是，此方法可用于同时处理多个更小的样品（1毫升或更小的细胞）。许多不同的有孔玻璃珠或珠磨机干扰矩阵现在市售扰乱几乎任何种类的细胞类型在2ml管。考虑到其他的技术和设备，珠磨机具有额外的好处，酵母细胞中的中断发生得非常快，从而有助于保持翻译后修饰，如SUMO化的，尤其是当使用蛋白酶和/或蛋白酶抑制剂与合适的缓冲液提取物的温度控制。 该协议的重点在温和的条件下，其最终目标保持蛋白质的功能，相互作用和翻译后修饰的快速，有效，可靠的内源性和过度表达的蛋白质提取。生长培养基，细胞裂解液组合物，珠磨机设置优化，保持蛋白质相互作用和SUMO化的和泛素化等翻译后修饰。

<table border="0" cellpadding="0" cellspacing="0" width="1039">
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	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Omni Bead Ruptor 24</td>
			<td style="width:124px;">Omni International</td>
			<td style="width:119px;">19-010</td>
			<td style="width:580px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Yeast ORF strain in BG1805</td>
			<td>ThermoScientific</td>
			<td>YSC3869</td>
			<td>GAL+ yeast strain that can be used for induction and extraction</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">pYES2.1 TOPO vector</td>
			<td>Life Technologies</td>
			<td>K4150-01</td>
			<td>GAL+ yeast strain that can be used for induction and extraction; contains a V5/6xHIS tag</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Halt Protease Inhibitor Single-Use Cocktail (100x)</td>
			<td>Thermo Scientific</td>
			<td style="width:119px;">1860932</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">2.0 ml Skirted Tube with Tethered Screw Cap</td>
			<td>BioExpress</td>
			<td style="width:119px;">C-3369-3</td>
			<td>Make sure that the tube properly fits in the bead ruptor</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Glass beads, acid-washed, 425-600 &#181;m (30-40 US sieve)</td>
			<td>Sigma-Aldrich</td>
			<td>G8772</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Corning Costar Spin-X centrifuge tube filters</td>
			<td>Sigma-Aldrich</td>
			<td style="width:119px;">CLS8161</td>
			<td>Use for additional filtering of clarified lysate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TALON Metal Affinity Resin</td>
			<td style="width:124px;">Clontech</td>
			<td>635502</td>
			<td>For the purification of 6xHIS tagged proteins</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">NuPAGE LDS Sample Buffer (4x)</td>
			<td style="width:124px;">Life Technologies</td>
			<td>NP0007</td>
			<td>To prepare and/or elute samples prior to SDS-PAGE and Western Blotting</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NuPAGE 4-12% Bis-Tris Gel</td>
			<td style="width:124px;">Life Technologies</td>
			<td style="width:119px;">NP0321BOX</td>
			<td>Precast gels often used for SDS-PAGE prior to Western Blotting</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Simply Blue</td>
			<td style="width:124px;">Life Technologies</td>
			<td style="width:119px;">#LC6060</td>
			<td>Protein gel stain</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Mammalian Lysis Buffer</td>
			<td>Promega</td>
			<td style="width:119px;">G9381</td>
			<td>Alternative commercial lysis buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Anti-V5 agarose</td>
			<td>Sigma</td>
			<td style="width:119px;">A7345</td>
			<td>Method of immunoprecipitation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">ECL</td>
			<td style="width:124px;">Millipore</td>
			<td style="width:119px;">WBKL S0 050</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">PVDF membrane</td>
			<td style="width:124px;">Millipore</td>
			<td>IPVH00010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">BIS-TRIS gels</td>
			<td style="width:124px;">Life Technologies</td>
			<td>NP0321BOX</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">anti-myc antibody</td>
			<td style="width:124px;">Covance</td>
			<td style="width:119px;">mms-150R</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">secondary antibody</td>
			<td style="width:124px;">Abcam</td>
			<td style="width:119px;">ab97040</td>
			<td>Goat pAb to mouse IgG (HRP)</td>
		</tr>
	</tbody>
</table>

budding yeast protein extraction and purification for the study of function, interactions, and post-translational modifications

珠跳动的同质化是一个快速和有效的方式来释放DNA，RNA，蛋白质和代谢产物，从芽殖酵母细胞，这是出了名的难以破坏。在这里，我们描述了使用珠磨机，均化器优化，保持的功能，相互作用的蛋白质的翻译后修饰的蛋白质的提取到缓冲区。对数生长期的细胞表达的蛋白质还给出的液体生长介质中生长。生长培养基可以补充试剂诱导蛋白表达诱导型启动子（ 如半乳糖），细胞周期阶段的同步（ 例如，诺考达唑），或抑制蛋白酶体的功能（ 例如，蛋白酶体抑制剂MG132）。细胞再沉淀，再悬浮在适当的缓冲液含有蛋白酶和/或磷酸酶抑制剂，被立即处理，或在液氮中冷冻，以供稍后使用。同质化是通过6个周期的20秒BEA的d的跳动（5.5米/秒），然后在冰上保温一分钟。通过离心分离所得到的匀浆被清除，小颗粒可通过过滤除去。由此产生的清除的全细胞提取物（WCE）沉淀，用20％三氯乙酸的总蛋白，通过SDS-PAGE，然后通过Western印迹法直接分析。提取物也适合用于亲和纯化的特定蛋白质的翻译后修饰，或共同净化蛋白的分析，检测。大多数蛋白质的纯化协议的情况下，可能需要一些酶和蛋白质的独特条件或缓冲液的组合物的纯化和其他规定的条件下可能是不稳定的或不溶性的。在后者的情况下，提出的协议可提供一个有用的起点，凭经验确定最佳的蛋白提取和纯化珠跳动策略。我们显示了提取和纯化的表位标记的SUMO E3连接酶，SIZ1，一个细胞周期调节蛋白变得SUMO化和磷酸化，以及SUMO针对性的泛素连接酶亚基，Slx5。

我们的代表性的结果揭示了用于各种下游应用（总结在表2）的蛋白质的制备重现性，所描述的珠跳动和蛋白质提取协议是有用的。然后通过考马斯亮蓝染色的WCEs的SDS-PAGE表明，蛋白质（〜12&gt; 250 kDa）的广泛的可再现性地从酵母细胞中（ 图1A）萃取。分子量在一定范围内的离散频段的优质蛋白质提取物的指标。提取物的质量可以针对特定抗原标记的蛋白质免疫印迹证实。...

Watch this Scientific Journal Video about Budding Yeast Protein Extraction and Purification for the Study of Function, Interactions, and Post-translational Modifications at JoVE.com

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

高品质的酵母细胞提取物的制备是一个必要的分析单个蛋白质或整个蛋白质组的第一步。在这里，我们描述了一个快速，高效，可靠的同质化协议已被优化，以保持蛋白质的功能，相互作用和翻译后修饰的芽殖酵母细胞。

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 ...

budding-yeast-protein-extraction-purification-for-study-function

Research

JoVE Journal

Biology

24.8K 次观看.  The College of William & Mary. 高品质的酵母细胞提取物的制备是一个必要的分析单个蛋白质或整个蛋白质组的第一步。在这里，我们描述了一个快速，高效，可靠的同质化协议已被优化，以保持蛋白质的功能，相互作用和翻译后修饰的芽殖酵母细胞。 

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

Measuring Replicative Life Span in the Budding Yeast

Aging is a degenerative process characterized by a progressive deterioration of cellular components and organelles resulting in mortality. The budding yeast Saccharomyces cerevisiae has been used extensively to study the biology of aging, and several determinants of yeast longevity have been shown to be conserved in multicellular eukaryotes, including worms, flies, and mice 1. Due to the lack of easily quantified age-associated phenotypes, aging in yeast has been assayed almost exclusively by measuring the life span of cells in different contexts, with two different life span paradigms in common usage 2. Chronological life span refers to the length of time that a mother cell can survive in a non-dividing, quiescence-like state, and is proposed to serve as a model for aging of post-mitotic cells in multicellular eukaryotes. Replicative life span, in contrast, refers the number of daughter cells produced by a mother cell prior to senescence, and is thought to provide a model of aging in mitotically active cells. Here we present a generalized protocol for measuring the replicative life span of budding yeast mother cells. The goal of the replicative life span assay is to determine how many times each mother cell buds. The mother and daughter cells can be easily differentiated by an experienced researcher using a standard light microscope (total magnification 160X), such as the Zeiss Axioscope 40 or another comparable model. Physical separation of daughter cells from mother cells is achieved using a manual micromanipulator equipped with a fiber-optic needle. Typical laboratory yeast strains produce 20-30 daughter cells per mother and one life span experiment requires 2-3 weeks.

In this article we present a general protocol for measuring the replicative life span of yeast mother cells.

在芽殖酵母中的自我复制寿命测量

Aging is a degenerative process characterized by a progressive deterioration of cellular components and organelles resulting in mortality. The budding ...

科研

生物学

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 ...

Analysis of the Development of a Morphological Phenotype as a Function of Protein Concentration in Budding Yeast

Gene deletion and protein overexpression are common methods for studying functions of proteins. In this article, we describe a protocol for analysis of phenotype development as a function of protein concentration at population and single-cell levels in Saccharomyces cerevisiae. Although this protocol is based on the overexpression of a protein, it can easily be adapted for morphological phenotypes dependent on suppression of protein expression. Our lab is interested in studying the signaling properties of the endocytic adaptor protein epsin. To that purpose we used a dominant negative approach in which we over-expressed the conserved Epsin N-Terminal Homology (ENTH) domain in order to interfere with the functions of endogenous epsin-2 (Ent2 or YLR206W). We observed that overexpression of the ENTH domain of Ent2 (ENTH2) in wild type cells led to a cell division defect that is dependent on the mislocalization of a family of scaffolding proteins, septins.

Gene deletion and protein overexpression are common methods for studying functions of proteins. In this article, we describe a protocol for analysis of phenotype development as a function of protein concentration at population and single-cell levels in Saccharomyces cerevisiae.

在芽殖酵母中的蛋白浓度的功能形态学表型的发展分析

Gene deletion and protein overexpression are common methods for studying functions of proteins. In this article, we describe a protocol for analysis of ...

Detection of Post-translational Modifications on Native Intact Nucleosomes by ELISA

The genome of eukaryotes exists as chromatin which contains both DNA and proteins. The fundamental unit of chromatin is the nucleosome, which contains 146 base pairs of DNA associated with two each of histones H2A, H2B, H3, and H41. The N-terminal tails of histones are rich in lysine and arginine and are modified post-transcriptionally by acetylation, methylation, and other post-translational modifications (PTMs). The PTM configuration of nucleosomes can affect the transcriptional activity of associated DNA, thus providing a mode of gene regulation that is epigenetic in nature 2,3. We developed a method called nucleosome ELISA (NU-ELISA) to quantitatively determine global PTM signatures of nucleosomes extracted from cells. NU-ELISA is more sensitive and quantitative than western blotting, and is useful to interrogate the epiproteomic state of specific cell types. This video journal article shows detailed procedures to perform NU-ELISA analysis.

Nucleosome ELISA (NU-ELISA) is a sensitive and quantitative method to detect global patterns of post-translational modifications in preparations of native, intact nucleosomes. These modifications include methylations, acetylations, and phosphorylations at specific histone amino acid residues, and hence NU-ELISA provides a global proteomic assay of the overall chromatin modification states of specific cell types.

检测采用ELISA法的母语完整的核小体的翻译后修饰

The genome of eukaryotes exists as chromatin which contains both DNA and proteins. The fundamental unit of chromatin is the nucleosome, which contains 146 ...

Identification of Post-translational Modifications of Plant Protein Complexes

Plants adapt quickly to changing environments due to elaborate perception and signaling systems. During pathogen attack, plants rapidly respond to infection via the recruitment and activation of immune complexes. Activation of immune complexes is associated with post-translational modifications (PTMs) of proteins, such as phosphorylation, glycosylation, or ubiquitination. Understanding how these PTMs are choreographed will lead to a better understanding of how resistance is achieved.
Here we describe a protein purification method for nucleotide-binding leucine-rich repeat (NB-LRR)-interacting proteins and the subsequent identification of their post-translational modifications (PTMs). With small modifications, the protocol can be applied for the purification of other plant protein complexes. The method is based on the expression of an epitope-tagged version of the protein of interest, which is subsequently partially purified by immunoprecipitation and subjected to mass spectrometry for identification of interacting proteins and PTMs.
This protocol demonstrates that: i). Dynamic changes in PTMs such as phosphorylation can be detected by mass spectrometry; ii). It is important to have sufficient quantities of the protein of interest, and this can compensate for the lack of purity of the immunoprecipitate; iii). In order to detect PTMs of a protein of interest, this protein has to be immunoprecipitated to get a sufficient quantity of protein.

We describe here a protocol for the purification and characterization of plant protein complexes. We demonstrate that by immunoprecipitating a single protein within a complex, so we can identify its post-translational modifications and its interacting partners.

植物蛋白复合体的翻译后修饰的鉴定

Plants adapt quickly to changing environments due to elaborate perception and signaling systems. During pathogen attack, plants rapidly respond to ...

Combining Single-molecule Manipulation and Imaging for the Study of Protein-DNA Interactions

The paper describes the combination of optical tweezers and single molecule fluorescence detection for the study of protein-DNA interaction. The method offers the opportunity of investigating interactions occurring in solution (thus avoiding problems due to closeby surfaces as in other single molecule methods), controlling the DNA extension and tracking interaction dynamics as a function of both mechanical parameters and DNA sequence. The methods for establishing successful optical trapping and nanometer localization of single molecules are illustrated. We illustrate the experimental conditions allowing the study of interaction of lactose repressor (lacI), labeled with Atto532, with a DNA molecule containing specific target sequences (operators) for LacI binding. The method allows the observation of specific interactions at the operators, as well as one-dimensional diffusion of the protein during the process of target search. The method is broadly applicable to the study of protein-DNA interactions but also to molecular motors, where control of the tension applied to the partner track polymer (for example actin or microtubules) is desirable.

Here we describe the instrumentation and methods for detecting single fluorescently-labeled protein molecules interacting with a single DNA molecule suspended between two optically trapped microspheres.

结合单分子操纵和成像蛋白-DNA相互作用的研究

The paper describes the combination of optical tweezers and single molecule fluorescence detection for the study of protein-DNA interaction. The method ...

Discovering Protein Interactions and Characterizing Protein Function Using HaloTag Technology

Research in proteomics has exploded in recent years with advances in mass spectrometry capabilities that have led to the characterization of numerous proteomes, including those from viruses, bacteria, and yeast.&#160; In comparison, analysis of the human proteome lags behind, partially due to the sheer number of proteins which must be studied, but also the complexity of networks and interactions these present. To specifically address the challenges of understanding the human proteome, we have developed HaloTag technology for protein isolation, particularly strong for isolation of multiprotein complexes and allowing more efficient capture of weak or transient interactions and/or proteins in low abundance. &#160;HaloTag is a genetically encoded protein fusion tag, designed for covalent, specific, and rapid immobilization or labelling of proteins with various ligands. Leveraging these properties, numerous applications for mammalian cells were developed to characterize protein function and here we present methodologies including: protein pull-downs used for discovery of novel interactions or functional assays, and cellular localization. We find significant advantages in the speed, specificity, and covalent capture of fusion proteins to surfaces for proteomic analysis as compared to other traditional non-covalent approaches. We demonstrate these and the broad utility of the technology using two important epigenetic proteins as examples, the human bromodomain protein BRD4, and histone deacetylase HDAC1.&#160; These examples demonstrate the power of this technology in enabling &#160;the discovery of novel interactions and characterizing cellular localization in eukaryotes, which will together further understanding of human functional proteomics. &#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;

HaloTag technology is a multifunctional technology which has shown significant success in isolation of both small and large protein complexes from mammalian cells.&#160; Here we highlight the advantages of this technology compared to existing alternatives and demonstrate its utility to study numerous aspects of protein function inside eukaryotic cells.

发现蛋白质相互作用和检定蛋白质功能使用的HaloTag技术

Research in proteomics has exploded in recent years with advances in mass spectrometry capabilities that have led to the characterization of numerous ...

Protein Purification Technique that Allows Detection of Sumoylation and Ubiquitination of Budding Yeast Kinetochore Proteins Ndc10 and Ndc80

Post-translational Modifications (PTMs), such as phosphorylation, methylation, acetylation, ubiquitination, and sumoylation, regulate the cellular function of many proteins. PTMs of kinetochore proteins that associate with centromeric DNA mediate faithful chromosome segregation to maintain genome stability. Biochemical approaches such as mass spectrometry and western blot analysis are most commonly used for identification of PTMs. Here, a protein purification method is described that allows the detection of both sumoylation and ubiquitination of the kinetochore proteins, Ndc10 and Ndc80, in Saccharomyces cerevisiae. A strain that expresses polyhistidine-Flag-tagged Smt3 (HF-Smt3) and Myc-tagged Ndc10 or Ndc80 was constructed and used for our studies. For detection of sumoylation, we devised a protocol to affinity purify His-tagged sumoylated proteins by using nickel beads and used western blot analysis with anti-Myc antibody to detect sumoylated Ndc10 and Ndc80. For detection of ubiquitination, we devised a protocol for immunoprecipitation of Myc-tagged proteins and used western blot analysis with anti-Ub antibody to show that Ndc10 and Ndc80 are ubiquitinated. Our results show that epitope tagged-protein of interest in the His-Flag tagged Smt3 strain facilitates the detection of multiple PTMs. Future studies should allow exploitation of this technique to identify and characterize protein interactions that are dependent on a specific PTM.

This manuscript describes the detection of sumoylation and ubiquitination of kinetochore proteins, Ndc10 and Ndc80, in the budding yeast Saccharomyces cerevisiae.

蛋白质纯化技术，可以检测SUMO化修饰和芽殖酵母蛋白动粒和Ndc10泛素Ndc80的

Post-translational Modifications (PTMs), such as phosphorylation, methylation, acetylation, ubiquitination, and sumoylation, regulate the cellular ...

Complete Workflow for Analysis of Histone Post-translational Modifications Using Bottom-up Mass Spectrometry: From Histone Extraction to Data Analysis

Nucleosomes are the smallest structural unit of chromatin, composed of 147 base pairs of DNA wrapped around an octamer of histone proteins. Histone function is mediated by extensive post-translational modification by a myriad of nuclear proteins. These modifications are critical for nuclear integrity as they regulate chromatin structure and recruit enzymes involved in gene regulation, DNA repair and chromosome condensation. Even though a large part of the scientific community adopts antibody-based techniques to characterize histone PTM abundance, these approaches are low throughput and biased against hypermodified proteins, as the epitope might be obstructed by nearby modifications. This protocol describes the use of nano liquid chromatography (nLC) and mass spectrometry (MS) for accurate quantification of histone modifications. This method is designed to characterize a large variety of histone PTMs and the relative abundance of several histone variants within single analyses. In this protocol, histones are derivatized with propionic anhydride followed by digestion with trypsin to generate peptides of 5 - 20 aa in length. After digestion, the newly exposed N-termini of the histone peptides are derivatized to improve chromatographic retention during nLC-MS. This method allows for the relative quantification of histone PTMs spanning four orders of magnitude.

This protocol outlines a fully integrated workflow for characterizing histone post-translational modifications using mass spectrometry (MS). The workflow includes histone purification from cell cultures or tissues, histone derivatization and digestion, MS analysis using nano-flow liquid chromatography and instructions for data analysis. The protocol is designed for completion within 2 - 3 days.

完整的工作流程为组蛋白翻译后修饰的分析采用自下而上质谱法：从组蛋白提取数据分析

Nucleosomes are the smallest structural unit of chromatin, composed of 147 base pairs of DNA wrapped around an octamer of histone proteins. Histone ...

Applications of pHluorin for Quantitative, Kinetic and High-throughput Analysis of Endocytosis in Budding Yeast

Green fluorescent protein (GFP) and its variants are widely used tools for studying protein localization and dynamics of events such as cytoskeletal remodeling and vesicular trafficking in living cells. Quantitative methodologies using chimeric GFP fusions have been developed for many applications; however, GFP is somewhat resistant to proteolysis, thus its fluorescence persists in the lysosome/vacuole, which can impede quantification of cargo trafficking in the endocytic pathway. An alternative method for quantifying endocytosis and post-endocytic trafficking events makes use of superecliptic pHluorin, a pH-sensitive variant of GFP that is quenched in acidic environments. Chimeric fusion of pHluorin to the cytoplasmic tail of transmembrane cargo proteins results in a dampening of fluorescence upon incorporation of the cargo into multivesicular bodies (MVBs) and delivery to the lysosome/vacuole lumen. Thus, quenching of vacuolar fluorescence facilitates quantification of endocytosis and early events in the endocytic pathway. This paper describes methods using pHluorin-tagged cargos for quantification of endocytosis via fluorescence microscopy, as well as population-based assays using flow cytometry.

Accurate quantification of vesicular trafficking events often provides key insights into roles for specific proteins and the effects of mutations. This paper presents methods for using superecliptic pHluorin, a pH-sensitive GFP variant, as a tool for quantification of endocytic events in living cells using quantitative fluorescence microscopy and flow cytometry.

pHluorin的申请在芽殖酵母细胞内吞的定量，动力学和高通量分析

Green fluorescent protein (GFP) and its variants are widely used tools for studying protein localization and dynamics of events such as cytoskeletal ...