Name: budding yeast protein extraction and purification for the study of function, interactions, and post-translational modifications
Uploaded: 2013-10-30T22:35:00
Description: 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

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</...

<ol>
	<li>Klis, F. M., Boorsma, A., De Groot, P. W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+wall+construction+inSaccharomyces+cerevisiae.">Cell wall construction inSaccharomyces cerevisiae.</a> Yeast. 23 (3), 185-202 (2006).</li><li>Gelperin, D. M., White, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biochemical+and+genetic+analysis+of+the+yeast+proteome+with+a+movable+ORF+collection.">Biochemical and genetic analysis of the yeast proteome with a movable ORF collection.</a> Genes Dev. 19 (23), 2816-2826 (2005).</li><li>Caserta, E., Haemig, H. A. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+Vivo+and+In+Vitro+Analyses+of+Regulation+of+the+Pheromone-Responsive+prgQ+Promoter+by+the+PrgX+Pheromone+Receptor+Protein.">In Vivo and In Vitro Analyses of Regulation of the Pheromone-Responsive prgQ Promoter by the PrgX Pheromone Receptor Protein.</a> J. Bacteriol. 194 (13), 3386-3394 (2012).</li><li>Ghaemmaghami, S., Huh, W. -. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+analysis+of+protein+expression+in+yeast.">Global analysis of protein expression in yeast.</a> Nature. 425 (6959), 737-741 (2003).</li><li>Hannich, J. T., Lewis, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defining+the+SUMO-modified+proteome+by+multiple+approaches+in+Saccharomyces+cerevisiae.">Defining the SUMO-modified proteome by multiple approaches in Saccharomyces cerevisiae.</a> J. Biol. Chem. 280 (6), 4102-4110 (2005).</li><li>Xie, Y., Rubenstein, E. M., Matt, T., Hochstrasser, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SUMO-independent+in+vivo+activity+of+a+SUMO-targeted+ubiquitin+ligase+toward+a+short-lived+transcription+factor.">SUMO-independent in vivo activity of a SUMO-targeted ubiquitin ligase toward a short-lived transcription factor.</a> Genes Dev. 24 (9), 893-903 (2010).</li><li>Daum, G. G., B&#246;hni, P. C. P., Schatz, G. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Import+of+proteins+into+mitochondria.+Cytochrome+b2+and+cytochrome+c+peroxidase+are+located+in+the+intermembrane+space+of+yeast+mitochondria.">Import of proteins into mitochondria. Cytochrome b2 and cytochrome c peroxidase are located in the intermembrane space of yeast mitochondria.</a> J. Biol. Chem. 257 (21), 13028-13033 (1982).</li><li>Deutscher, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Guide to protein purification. , (1990).</li><li>Puig, O., Caspary, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Tandem+Affinity+Purification+(TAP)+Method:+A+General+Procedure+of+Protein+Complex+Purification.">The Tandem Affinity Purification (TAP) Method: A General Procedure of Protein Complex Purification.</a> Methods. 24 (3), 218-229 (2001).</li><li>Conzelmann, A., Riezman, H., Desponds, C., Bron, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+major+125-kd+membrane+glycoprotein+of+Saccharomyces+cerevisiae+is+attached+to+the+lipid+bilayer+through+an+inositol-containing+phospholipid.">A major 125-kd membrane glycoprotein of Saccharomyces cerevisiae is attached to the lipid bilayer through an inositol-containing phospholipid.</a> EMBO J. 7 (7), 2233 (1988).</li><li>Dertzbaugh, M. T. M., Cox, L. M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+affinity+of+cholera+toxin+for+Ni2++ion.">The affinity of cholera toxin for Ni2+ ion.</a> Protein Eng. 11 (7), 577-581 (1998).</li><li>Hautbergue, G. G., Goguel, V. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+yeast+C-type+cyclin+Ctk2p+is+phosphorylated+and+rapidly+degraded+by+the+ubiquitin-proteasome+pathway.">The yeast C-type cyclin Ctk2p is phosphorylated and rapidly degraded by the ubiquitin-proteasome pathway.</a> Mol. Cell. Biol. 19 (4), 2527-2534 (1999).</li><li>Elmore, Z. C., Donaher, M., Matson, B. C., Murphy, H., Westerbeck, J. W., Kerscher, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sumo-dependent+substrate+targeting+of+the+SUMO+protease+Ulp1.">Sumo-dependent substrate targeting of the SUMO protease Ulp1.</a> BMC Biol. 9, 74 (2011).</li><li>Xie, Y., Kerscher, O., Kroetz, M. B., McConchie, H. F., Sung, P., Hochstrasser, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+yeast+Hex3.Slx8+heterodimer+is+a+ubiquitin+ligase+stimulated+by+substrate+sumoylation.">The yeast Hex3.Slx8 heterodimer is a ubiquitin ligase stimulated by substrate sumoylation.</a> J. Biol. Chem. 282 (47), 34176-34184 (2007).</li></ol>

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">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<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 ...

Research

JoVE Journal

Biology

Measuring Replicative Life Span in the Budding Yeast

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

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

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

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

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

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

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

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

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

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 ...