怒江- ELISA法的发展是支持由美国国立卫生研究院资助RO1AG023687。

1。哺乳动物细胞培养 NU - ELISA法可以在任何可以在培养的哺乳动物细胞类型。我们宁愿准备适度大批量的细胞，使核小体可以在足够数量的隔离准备几个相同加载的酶标板，从而使与几个不同的抗体（ABS）的检测。下面的文化尺度提供了充足的材料: 对于小鼠embyronic的干细胞，无饲养层细胞生长的细胞的一个或两个15厘米板。对于成纤维细胞，生长5年至10 15厘米板汇合。 2。原子核的隔离注:所有步骤都是冰预冷的缓冲，除特别说明外。 <ol><li> Trypsinize细胞，每盘用3 ml胰蛋白酶结合，并添加20毫升的冰冷的PBS /丁酸。离心5分钟1000转。 </li><li>重悬细胞于10 ml PBS /丁酸和离心5分钟1000转。 </li><li>悬浮在与蛋白酶抑制剂（Sigma - Aldrich公司的P - 8340）的细胞裂解液4毫升。 Dounce均质型碓冰乙20招。 </li><li>在2000克离心10分钟，在4 ° C。 （上清含有细胞质，这是不是本议定书所需要的，但如果需要的话，可以作其他用途，保存）。 </li><li>悬浮颗粒在2毫升冰冷洗缓冲液C（蛋白酶抑制剂）。 </li><li>层超过500毫升30％的蔗糖垫的悬浮物质，然后在2400克离心5分钟在一个水平转头。原子核会迁移，通过垫，接口仍然碎片。 </li><li>仔细删除所有液体的体积和重悬在250μL冰冷的缓冲液洗的C +蛋白酶抑制剂的原子核。 </li></ol> 3。核小体的分离在原位微球菌核酸（MNase）消化我们使用在其中染色质内的细胞核注入与MNase，由低渗处理上清液，从而推动了免费的完整的核小体原位消化的过程。 <ol><li> 3μL0.1 M的氯化钙样品。放置在37℃加热块。允许承担37 ° C的温度。 </li><li>新增2台MNase（Sigma - Aldrich公司，微球菌核酸，溶解于2units/10μlMNase缓冲区），然后在37 ° C为12分钟，经常混合使用吸管尖。 </li><li>加入6μL0.5 M的Na - EDTA，pH值8.0，停止反应。置于冰上。 </li><li>在2000克离心4分钟，弃上清。 300μL0.2毫米的Na - EDTA重悬沉淀。存放在冰与偶尔的温柔pipeting 1小时。 （这些低渗条件，从原子核中解放出来的自由核小体）。 </li><li>离心4分钟3000克，在4 ° C。保存上清，其中包含了免费的核小体，在冰上。 </li><li>悬浮颗粒再次与300μL0.2毫米的Na - EDTA。存放在冰与偶尔的温柔pipeting 1小时。 </li><li>离心4分钟3000克在4 ° C。保留上清，结合起来，从第5步上清。造成mononucleosome准备可以分装和储存在-80 ° C </li></ol>注:染色质量可以粗略地通过测量10μL样品在260 nm处的吸光度定量加入到990μL水。 260 = 10（后调整为1 / 100稀释），相当于约染色1mg/ml的。此外，在上述过程中，小样本可能被保留，并为他们的DNA含量分析后监察MNase消化，这主要应包含长度为146 bp的DNA的质量和程度。阶梯是MNase消化不全的指标。 注:图1包含一个核隔离和MNase消化步骤图解摘要。 4。核小体，ELISA（NU - ELISA法） 我们发现含有本机已在96孔酶标微孔板固定完好nuclesosomes分数PTMS。对于每个样本，我们做了一系列的2倍稀释，这些都是到涂井一式三份。 <ol><li>在4 ° 50μL/孔的核小体在涂料缓冲稀释过夜大衣MaxiSorp板建议核小体的序列双重稀释准备从最上面一行的底部行中加入0.1微克，0.05微克，0.025微克，0.0125微克，0.00625微克，0.00313微克，0.00156微克，与涂层的缓冲区，只有在底部（0微克染色）行。 （注:板盖都需要这一步。） </li><li>当天上午，洗板200μL/ PBS/0.5％吐温20在室温（RT）的总和为10分钟用洗板机的4倍。 </li><li>座在室温下1小时与100μL/ PBS/0.05％吐温20 / 5％牛血清白蛋白。 </li><li>删除轻快地摇晃倒板封闭液在一个水槽。盖上盖子，并储存在-20 ° C的板，或直接进入下一步。 </li><li> 1 · ABS添加量在50μL/ 1:1000以及稀释PBS/0.05％吐温20 / 5％BSA，室温下孵育1小时一个肩（或需要）。 </li><li>洗净板200μL/ PBS/0.5％吐温20在室温下，在洗板的总和为10分钟的4倍。 </li><li>加入辣根过氧化物酶（HRP）的共轭2 ° ABS，PBS/0.05％吐温20 / 5％BSA室温下一个小时一个肩稀释1:5000（或需要）。 </li><li>洗净板与200μL/孔PBS/0.5％Tween - 20的4倍。每次清洗用洗板机在室温下，共10分钟。 </li><li>在室温为10分钟，加入50μLTMB底物，每口井的开发板。加入50μL/孔的2N H 2 SO 4，停止反应，450 nm处读取的板块。 （提示:2分钟1500转的离心机，使用一盘转子任何气泡​​消散在井前读取吸光度）。 </li><li>读数导出到电子表格文件，定量和统计分析。 </li></ol>试剂<table border="1"><tbody><tr><td> PBS /丁酸 </td></tr><tr><td> 135 mM氯化钠</td></tr><tr><td> 2.5 mM的氯化钾</td></tr><tr><td> 8毫米的Na 2 HPO 4 </td></tr><tr><td> 1.5毫米KH 2 PO 4 </td></tr><tr><td> 10毫米钠丁酸</td></tr></tbody></table><table border="1"><tbody><tr><td> 裂解液 </td></tr><tr><td> 250毫米蔗糖</td></tr><tr><td> 10毫米的Tris - HCl，pH值7.4 </td></tr><tr><td> 10毫米钠丁酸</td></tr><tr><td> 4毫米氯化镁2 </td></tr><tr><td> 0.1％的Triton X - 100 </td></tr></tbody></table><table border="1"><tbody><tr><td> 洗缓冲液C </td></tr><tr><td> 250毫米蔗糖</td></tr><tr><td> 10毫米的Tris - HCl，pH值7.4 </td></tr><tr><td> 10毫米钠丁酸</td></tr><tr><td> 4毫米氯化镁2 </td></tr></tbody></table><table border="1"><tbody><tr><td> 蔗糖垫 </td></tr><tr><td> 30％（W / V）蔗糖在洗涤缓冲液C </td></tr></tbody></table><table border="1"><tbody><tr><td> Microccocal核酸缓冲器 </td></tr><tr><td> 5毫米NAPO 4缓冲液，pH值7.0 </td></tr><tr><td> 0.025毫米氯化钙2 </td></tr></tbody></table><table border="1"><tbody><tr><td> 涂料缓冲区 </td></tr><tr><td> 80毫升溶液A +170毫升解决方案的B +250毫升生署2 O </td></tr><tr><td>溶液A:0.2 M NA 2 CO 3 </td></tr><tr><td> B液:0.2 M碳酸氢钠3 </td></tr></tbody></table> 5。代表性的成果使用NU - ELISA检测方法，准备与染色质的mononucleosomes形式编写的，它可以载入多个相同的板系列。我们通常会准备一系列的相同装板，使每个人都可以用不同的反PTM（ABS）的特异性抗体探测。我们也随时准备与组蛋白抗体，检测，而不考虑其修改状态总染色质负荷控制，可探测的一个板块。这种控制是必不可少的，为了以后定量PTM的内容从不同的样品制备的核小体进行比较。在一个给定的染色质样品进行定量PTMS的水平，我们正确的原始吸光度值，使用这种方法:首先，我们使用不包含染色质（背景通常是可以忽略不计）从控制井获得的值减去任何背景信号。然后，我们规范的PTM -特定的信号相同装板与独立对其进行修改状态检测核小体的AB检测所得的NU - ELISA法检测的结果。 （我们用于此目的的组蛋白H2A或H2B多克隆ABS）。然后，我们决定为每个稀释的染色质的均值和方差，并用ELISA法检测的线性部分获得的数据。 NU - ELISA法的数学和统计分析的详细的例子有报道先前 4 <img src="/files/ftp_upload/2593/2593fig1.jpg" alt="图1"/> 图1。 NU - ELISA的步骤示意图diagam。 A.哺乳动物细胞的收获; B.细胞核从细胞中分离Dounce homogenization，C.扰乱细胞的30％W / V蔗糖垫，D.离心后的上加载，原子核的颗粒沉积在E，F染色质主要由MNase mononucleomes原位消化，G， H ，我提取可溶性mononucleosomes低渗处理和反复离心剩余的核材料。研究Mononucleosomes从不同的样本（标记桑普在这种情况下，1和2）涂在酶标井系列相同装板和审讯PTM - ABS和PTM -独立的ABS，确定总的染色质加载。K.差分信号的描述在他们的PTM内容不同的两个样品的检测水平，尚未有类似的整体染色质的内容，如反H2B免疫反应来判断。

<ol>
	<li>Luger, K., Mader, A. W., Richmond, R. K., Sargent, D. F., Richmond, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystal+structure+of+the+nucleosome+core+particle+at+2.8+A+resolution.">Crystal structure of the nucleosome core particle at 2.8 A resolution.</a> Nature. 389, 251-260 (1997).</li><li>Jenuwein, T., Allis, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Translating+the+histone+code.">Translating the histone code.</a> Science. 293, 1074-1080 (2001).</li><li>Turner, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histone+acetylation+and+an+epigenetic+code.">Histone acetylation and an epigenetic code.</a> Bioessays. 22, 836-845 (2000).</li><li>Dai, B., Rasmussen, T. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+epiproteomic+signatures+distinguish+embryonic+stem+cells+from+differentiated+cells.">Global epiproteomic signatures distinguish embryonic stem cells from differentiated cells.</a> Stem Cells. 25, 2567-2574 (2007).</li><li>Tanasijevic, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progressive+accumulation+of+epigen+etic+heterogeneity+during+human+ES+cell+culture.">Progressive accumulation of epigen etic heterogeneity during human ES cell culture.</a> Epigenetics. 4, 330-338 (2009).</li><li>Thomas, J. O., Gould, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+fractionation+of+chromatin+and+linker+histones.">Isolation and fractionation of chromatin and linker histones.</a> Chromatin: a practical approach. , 12-14 (1998).</li><li>Thorne, A. W., Cary, P. D., Crane-Robinson, C., Gould, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extraction+and+separation+of+core+histones+and+non-histone+chromosomal+proteins.">Extraction and separation of core histones and non-histone chromosomal proteins.</a> Chromatin: a practical approach. , 38-39 (1998).</li></ol>

NU - ELISA法检测提供了一种方法，以确定在一个特定的细胞类型中的核小体PTMS的全球的地位。 NU - ELISA法研究表明，全球核小体修饰国发散的细胞类型4比较不同。此外，NU - ELISA法的PTM配置文件的变化，当细胞暴露后生的调节剂如曲古抑菌素A或小鼠embyronic的干细胞分化 4时。该方法也被成功地应用于研究人类胚胎干细胞的染色质 5 。重要的是要注意的NU - ELISA法，以其目前的形式，可以检测只有在细胞表观总和目前PTMS复合签名。这明显不同于全基因组的染色质免疫沉淀，这可确定一个具体的PTM跨特定的基因位点的分布。 NU - ELISA法检测程序的最初步骤，是适应从以前的方法，从哺乳动物的细胞核6,7隔离mononucleosomes。这些适应的方法提供了一个权宜之计，从哺乳动物细胞中获得高品质的完整的mononucleosomes，以及由此产生的分数是在他们的染色质的内容全面，而且包含大量额外的核材料。由于特定的绝对值，污染，核小体材料的耐受性以及在怒江- ELISA法检测，不像大众规格的办法，需要更大的纯度。然而，NU - ELISA法更敏感，比定量免疫印迹，从而提供了很好的替代品西部片的核小体PTMS的检测和质量规范。

<table border="1"><tbody><tr><th>试剂名称</th><th>公司</th><th>目录编号</th><th>评论</th></tr><tr><td>微球菌Nucleae </td><td>西格玛</td><td> N3755 </td><td>等分2U/10μl缓冲区，存放在-20 ° C </td></tr><tr><td> NUNC MaxiSorp微孔板</td><td>费舍尔</td><td> 12-565-135 </td><td>胶粘板盖也可以通过费舍尔订购</td></tr><tr><td>自动洗板机</td><td></td><td></td><td></td></tr><tr><td> 1步TMB - ELISA基板</td><td>刺穿</td><td> 34028 </td><td>保存在4 ° C预温至RT，在使用前或规格表上的指示</td></tr><tr><td>酶标仪</td><td> BIO - RAD酶标仪（型号:680） </td><td></td><td>大多数比色法酶标仪模型适合</td></tr></tbody></table>

detection of post-translational modifications on native intact nucleosomes by elisa

真核生物的基因组中存在其中包含的DNA和蛋白质的染色。染色质的基本单位是核小体，其中包含146个DNA碱基对与两个组蛋白H2A，H2B，H3和H4 1相关的。组蛋白的N -末端尾巴中含有丰富的赖氨酸和精氨酸和转录后进行修改，经乙酰化，甲基化和其他翻译后修饰（PTMS）。 PTM的核小体配置会影响相关的DNA的转录活性，从 ​​而提供了一种基因调控的模式，是在自然界2,3后生。我们开发了一个方法叫做核小体酶联免疫吸附（NU - ELISA）定量测定，从细胞中提取的核小体的全球的PTM签名。 NU - ELISA法更为敏感和免疫印迹定量，并询问epiproteomic国家特定的细胞类型是有用的。此视频杂志文章显示详细程序执行NU - ELISA法分析。

Watch this Scientific Journal Video about Detection of Post-translational Modifications on Native Intact Nucleosomes by ELISA at JoVE.com

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

核小体酶联免疫吸附（NU - ELISA法）是一个敏感的和定量的方法来检测翻译后修饰的全球格局，本土的，完整的核小体的筹备工作。这些修改包括甲基化，acetylations，以及在特定的组蛋白的氨基酸残基的磷酸化，因此NU - ELISA法提供了一个全球性的整体染色修改国家特定的细胞类型的蛋白质组检测。

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 ...

detection-post-translational-modifications-on-native-intact

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

17.2K 次观看.  Stanford University. 核小体酶联免疫吸附（NU - ELISA法）是一个敏感的和定量的方法来检测翻译后修饰的全球格局，本土的，完整的核小体的筹备工作。这些修改包括甲基化，acetylations，以及在特定的组蛋白的氨基酸残基的磷酸化，因此NU - ELISA法提供了一个全球性的整体染色修改国家特定的细胞类型的蛋白质组检测。

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

Detection of Functional Matrix Metalloproteinases by Zymography

Matrix metalloproteinases (MMPs) are zinc-containing endopeptidases. They degrade proteins by cleavage of peptide bonds. More than twenty MMPs have been identified and are separated into six groups based on their structure and substrate specificity (collagenases, gelatinases, membrane type [MT-MMP], stromelysins, matrilysins, and others). MMPs play a critical role in cell invasion, cartilage degradation, tissue remodeling, wound healing, and embryogenesis. They therefore participate in both normal processes and in the pathogenesis of many diseases, such as rheumatoid arthritis, cancer, or chronic obstructive pulmonary disease1-6. Here, we will focus on MMP-2 (gelatinase A, type IV collagenase), a widely expressed MMP. We will demonstrate how to detect MMP-2 in cell culture supernatants by zymography, a commonly used, simple, and yet very sensitive technique first described in 1980 by C. Heussen and E.B. Dowdle7-10. This technique is semi-quantitative, it can therefore be used to determine MMP levels in test samples when known concentrations of recombinant MMP are loaded on the same gel11.
Solutions containing MMPs (e.g. cell culture supernatants, urine, or serum) are loaded onto a polyacrylamide gel containing sodium dodecyl sulfate (SDS; to linearize the proteins) and gelatin (substrate for MMP-2). The sample buffer is designed to increase sample viscosity (to facilitate gel loading), provide a tracking dye (bromophenol blue; to monitor sample migration), provide denaturing molecules (to linearize proteins), and control the pH of the sample. Proteins are then allowed to migrate under an electric current in a running buffer designed to provide a constant migration rate. The distance of migration is inversely correlated with the molecular weight of the protein (small proteins move faster through the gel than large proteins do and therefore migrate further down the gel). After migration, the gel is placed in a renaturing buffer to allow proteins to regain their tertiary structure, necessary for enzymatic activity. The gel is then placed in a developing buffer designed to allow the protease to digest its substrate. The developing buffer also contains p-aminophenylmercuric acetate (APMA) to activate the non-proteolytic pro-MMPs into active MMPs. The next step consists of staining the substrate (gelatin in our example). After washing the excess dye off the gel, areas of protease digestion appear as clear bands. The clearer the band, the more concentrated the protease it contains. Band staining intensity can then be determined by densitometry, using a software such as ImageJ, allowing for sample comparison.

This protocol describes an activity-based assay for detecting matrix metalloproteinases in culture supernatants or body fluids.

酶谱法检测功能的基质金属蛋白酶

Matrix metalloproteinases (MMPs) are zinc-containing endopeptidases. They degrade proteins by cleavage of peptide bonds. More than twenty MMPs have been ...

科研

生物学

Analyzing the Function of Small GTPases by Microinjection of Plasmids into Polarized Epithelial Cells

Epithelial cells polarize their plasma membrane into biochemically and functionally distinct apical and basolateral domains where the apical domain faces the 'free' surfaces and the basolateral membrane is in contact with the substrate and neighboring cells. Both membrane domains are separated by tight junctions, which form a diffusion barrier. Apical-basolateral polarization can be recapitulated successfully in culture when epithelial cells such as Madin-Darby Canine Kidney (MDCK) cells are seeded at high density on polycarbonate filters and cultured for several days 1 2. Establishment and maintenance of cell polarity is regulated by an array of small GTPases of the Ras superfamily such as RalA, Cdc42, Rab8, Rab10 and Rab13 3 4 5 6 7. Like all GTPases these proteins cycle between an inactive GDP-bound state and an active GTP-bound state. Specific mutations in the nucleotide binding regions interfere with this cycling 8. For example, Rab13T22N is permanently locked in the GDP-form and thus dubbed 'dominant negative', whereas Rab13Q67L can no longer hydrolyze GTP and is thus locked in a 'dominant active' state 7. To analyze their function in cells both dominant negative and dominant active alleles of GTPases are typically expressed at high levels to interfere with the function of the endogenous proteins 9. An elegant way to achieve high levels of overexpression in a short amount of time is to introduce the plasmids encoding the relevant proteins directly into the nuclei of polarized cells grown on filter supports using microinjection technique. This is often combined with the co-injection of reporter plasmids that encode plasma membrane receptors that are specifically sorted to the apical or basolateral domain. A cargo frequently used to analyze cargo sorting to the basolateral domain is a temperature sensitive allele of the vesicular stomatitis virus glycoprotein (VSVGts045) 10. This protein cannot fold properly at 39&deg;C and will thus be retained in the endoplasmic reticulum (ER) while the regulatory protein of interest is assembled in the cytosol. A shift to 31&deg;C will then allow VSVGts045 to fold properly, leave the ER and travel to the plasma membrane 11. This chase is typically performed in the presence of cycloheximide to prevent further protein synthesis leading to cleaner results. Here we describe in detail the procedure of microinjecting plasmids into polarized cells and subsequent incubations including temperature shifts that allow a comprehensive analysis of regulatory proteins involved in basolateral sorting.

This article details the procedures involved in overexpression and analysis of small GTPases in polarized epithelial cells using microinjection technique.

为极化上皮细胞注射质粒的小GTP酶的功能分析

Epithelial cells polarize their plasma membrane into biochemically and functionally distinct apical and basolateral domains where the apical domain faces ...

Detection of Histone Modifications in Plant Leaves

Chromatin structure is important for the regulation of gene expression in eukaryotes. In this process, chromatin remodeling, DNA methylation, and covalent modifications on the amino-terminal tails of histones H3 and H4 play essential roles1-2. H3 and H4 histone modifications include methylation of lysine and arginine, acetylation of lysine, and phosphorylation of serine residues1-2. These modifications are associated either with gene activation, repression, or a primed state of gene that supports more rapid and robust activation of expression after perception of appropriate signals (microbe-associated molecular patterns, light, hormones, etc.)3-7. 
Here, we present a method for the reliable and sensitive detection of specific chromatin modifications on selected plant genes. The technique is based on the crosslinking of (modified) histones and DNA with formaldehyde8,9, extraction and sonication of chromatin, chromatin immunoprecipitation (ChIP) with modification-specific antibodies9,10, de-crosslinking of histone-DNA complexes, and gene-specific real-time quantitative PCR. The approach has proven useful for detecting specific histone modifications associated with C4 photosynthesis in maize5,11 and systemic immunity in Arabidopsis3.

A reliable and useful approach to detect histone modifications on specific plant genes is described. The approach combines chromatin immunoprecipitation (ChIP) and real-time quantitative PCR. It allows detection of histone modifications on specific genes with a role in diverse physiological processes.

检测组蛋白修饰在植物叶片

Chromatin structure is important for the regulation of gene expression in eukaryotes. In this process, chromatin remodeling, DNA methylation, and covalent ...

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 ...

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 ...

Isolation of Specific Genomic Regions and Identification of Associated Molecules by enChIP

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the functions of these regions. To enable the non-biased identification of molecules interacting with a specific genomic region of interest, we recently developed the engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) technique. Here, we describe how to use enChIP to isolate specific genomic regions and identify the associated proteins and RNAs. First, a genomic region of interest is tagged with a transcription activator-like (TAL) protein or a clustered regularly interspaced short palindromic repeats (CRISPR) complex consisting of a catalytically inactive form of Cas9 and a guide RNA. Subsequently, the chromatin is crosslinked and fragmented by sonication. The tagged locus is then immunoprecipitated and the crosslinking is reversed. Finally, the proteins or RNAs that are associated with the isolated chromatin are subjected to mass spectrometric or RNA sequencing analyses, respectively. This approach allows the successful identification of proteins and RNAs associated with a genomic region of interest.

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the functions of these regions. This protocol describes procedures to perform engineered DNA-binding molecule-mediated chromatin imunoprecipitation (enChIP) for identification of proteins and RNAs associated with a specific genomic region.

具体的基因组区域及相关分子鉴定enChIP隔离

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the ...

En Face Detection of Nitric Oxide and Superoxide in Endothelial Layer of Intact Arteries

Endothelium-derived nitric oxide (NO) produced from endothelial NO-synthase (eNOS) is one of the most important vasoprotective molecules in cardiovascular physiology. Dysfunctional eNOS such as uncoupling of eNOS leads to decrease in NO bioavailability and increase in superoxide anion (O2.&#8722;) production, and in turn promotes cardiovascular diseases. Therefore, appropriate measurement of NO and O2.&#8722; levels in the endothelial cells are pivotal for research on cardiovascular diseases and complications. Because of the extremely labile nature of NO and O2.&#8722;, it is difficult to measure NO and O2.&#8722; directly in a blood vessel. Numerous methods have been developed to measure NO and O2.&#8722; production. It is, however, either insensitive, or non-specific, or technically demanding and requires special equipment. Here we describe an adaption of the fluorescence dye method for en face simultaneous detection and visualization of intracellular NO and O2.&#8722; using the cell permeable diaminofluorescein-2 diacetate (DAF-2DA) and dihydroethidium (DHE), respectively, in intact aortas of an obesity mouse model induced by high-fat-diet feeding. We could demonstrate decreased intracellular NO and enhanced O2.&#8722; levels in the freshly isolated intact aortas of obesity mouse as compared to the control lean mouse. We demonstrate that this method is an easy technique for direct detection and visualization of NO and O2.&#8722; in the intact blood vessels and can be widely applied for investigation of endothelial (dys)function under (physio)pathological conditions.

En Face Detection of Nitric Oxide and Superoxide in Endothelial Layer of Intact Arteries

In this article we describe an adapted relatively easy method using the fluorescence dye diaminofluorescein-2 diacetate (DAF-2DA) and dihydroethidium (DHE) for en face simultaneous detection and visualization of intracellular nitric oxide (NO) and superoxide anion (O2.&#8722;) respectively, in freshly isolated intact aortas of an obesity mouse model.

一氧化氮和超氧化物歧化酶恩的人脸检测在完整的动脉内皮细胞层

Endothelium-derived nitric oxide (NO) produced from endothelial NO-synthase (eNOS) is one of the most important vasoprotective molecules in cardiovascular ...

High-throughput CRISPR Vector Construction and Characterization of DNA Modifications by Generation of Tomato Hairy Roots

Targeted DNA mutations generated by vectors with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology have proven useful for functional genomics studies. While most cloning strategies are simple to perform, they generally use multiple steps and can require several days to generate the ultimate constructs. The method presented here is based on DNA assembly and can produce fully functional CRISPR vectors in a single cloning reaction. Vector construction can also be pooled, further increasing the efficiency and utility of the process. A modification of the method is used to create CRISPR vectors with multiple gene targets. CRISPR vectors are then transformed into tomato hairy roots to generate transgenic materials with targeted DNA modifications. Hairy roots are a useful system for testing vector functionality as they are technically simple to generate and amenable to large-scale production. The methods presented here will have wide application as they can be used to generate a variety of CRISPR vectors and be used in a wide range of plant species.

Using DNA assembly, multiple CRISPR vectors can be constructed in parallel in a single cloning reaction, making the construction of large numbers of CRISPR vectors a simple task. Tomato hairy roots are an excellent model system to validate CRISPR vectors and generate mutant materials.

高通量CRISPR载体的构建及DNA修饰的表征番茄毛状根的一代

Targeted DNA mutations generated by vectors with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology have proven useful for ...

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 ...

Comparison of Tobacco Host Cell Protein Removal Methods by Blanching Intact Plants or by Heat Treatment of Extracts

Plants not only provide food, feed and raw materials for humans, but have also been developed as an economical production system for biopharmaceutical proteins, such as antibodies, vaccine candidates and enzymes. These must be purified from the plant biomass but chromatography steps are hindered by the high concentrations of host cell proteins (HCPs) in plant extracts. However, most HCPs irreversibly aggregate at temperatures above 60 &#176;C facilitating subsequent purification of the target protein. Here, three methods are presented to achieve the heat precipitation of tobacco HCPs in either intact leaves or extracts. The blanching of intact leaves can easily be incorporated into existing processes but may have a negative impact on subsequent filtration steps. The opposite is true for heat precipitation of leaf extracts in a stirred vessel, which can improve the performance of downstream operations albeit with major changes in process equipment design, such as homogenizer geometry. Finally, a heat exchanger setup is well characterized in terms of heat transfer conditions and easy to scale, but cleaning can be difficult and there may be a negative impact on filter capacity. The design-of-experiments approach can be used to identify the most relevant process parameters affecting HCP removal and product recovery. This facilitates the application of each method in other expression platforms and the identification of the most suitable method for a given purification strategy.

Three heat precipitation methods are presented that effectively remove more than 90% of host cell proteins (HCPs) from tobacco extracts prior to any other purification step. The plant HCPs irreversibly aggregate at temperatures above 60 &#176;C.

通过热烫完整植株或提取物热处理烟草宿主细胞蛋白去除方法的比较

Plants not only provide food, feed and raw materials for humans, but have also been developed as an economical production system for biopharmaceutical ...