这项工作得到NIH R01 DK110355，DK093776 [NTS]，DK102450 [NTS]和P30 DK034987 [向UNC-Chapel Hill]的支持。作者感谢Deekshita Ramanarayanan协助qPCR和免疫印迹实验。

该协议是根据北卡罗来纳大学的机构动物保护和使用委员会（IACUC）批准和执行的。 准备工作<ol><li>制备Triton-X缓冲液（1％Triton X-100,5mM乙二胺四乙酸（EDTA）），在磷酸盐缓冲盐水（PBS），pH 7.4中调高体积）。制成500mL:将Triton X-100和500mM EDTA各5mL搅拌至490mL PBS（pH7.4）中。在4℃下储存Triton-X缓冲溶液。 </li><li>制备高盐缓冲液（10mM Tris-HCl，pH 7.6,140mM NaCl，1.5M KCl，5mM EDTA，0.5％Triton X-100，在双蒸馏（dd）H 2 O中调出体积）。制成500毫升:搅拌10mL 0.5M Tris-HCl（pH7.6），14mL 5M NaCl，55.9克KCl，5毫升0.5M EDTA和2.5毫升Triton X-100，将体积调至500 mL，使用双蒸水）。在4°C储存高盐缓冲溶液。 </li><li>在使用前，补充适量的Triton-X和高盐缓冲液<em&gt;例如，每25mg组织样品1mL）与蛋白酶或蛋白酶/磷酸酶抑制剂混合，并丢弃任何不含有抑制剂的缓冲液。 </li><li>在1×PBS，pH 7.4中制备5mM EDTA。制成500mL:将5mL的0.5M EDTA搅拌到495mL的1×PBS，pH7.4中。 </li><li>使用符合兽医指南和机构标准的认可方案，分离不同的小鼠器官（脑，心脏，肺，肝，胰腺，结肠，肠，肾，脾） 23 。组织采集程序应不超过5分钟，以保持富含蛋白酶的组织的RNA和蛋白质完整性（ 例如胰腺应先处理）。 </li><li>切割少量组织（〜5-20mg）并置于RNA储存溶液中，用于随后的RNA提取，cDNA合成和IF基因表达的定量实时PCR分析。将RNA储存溶液管与组织放置在4℃过夜，并遵循制造商协议进一步的折叠和隔离步骤。 </li><li>将其余的组织切成更小的碎片（ 例如 0.5厘米），并置于低温条件下。快速冷冻并将样品瓶储存在-80°C或液氮下长期储存。 </li></ol> IF基因表达分析<ol><li>从保存在RNA储存溶液试剂中的组织中提取RNA。根据制造商的方案使用任何合适/优选的RNA提取方法。 </li><li>定量RNA浓度，并使用2μgRNA根据制造商的方案使用合适的逆转录试剂盒产生cDNA。 </li><li>使用生成的cDNA和小鼠IF基因特异性引物设置qPCR反应，包括每个样品的三个技术重复以及根据制造商的方案的空白对照。 </li><li>将IF基因表达定量为比较不同条件（ 例如年轻组织和老组织）的折叠变化。 </li></ol> 3. P用于免疫印迹的总组织裂解物的修复<ol><li>在1mL的2x还原SDS样品缓冲液中均化25mg的组织。如果要进行比色蛋白测定以量化蛋白质浓度，则从样品缓冲液中除去溴酚蓝染料。 </li><li>加入5％（v / v）的2-巯基乙醇（2-ME）以制备还原样品。向200μL非还原样品中加入10μL2-ME。 </li><li>使用洗涤剂和还原剂相容的蛋白质测定法确定蛋白质浓度。如果染料已经包含在样品缓冲液中，蛋白质量可以通过许多技术运行凝胶之后估算，包括考马斯染色剂24 。 </li><li>涡旋所有样品并在95℃加热5分钟。 </li><li>在还原和非还原条件下进行Western印迹。短暂暴露膜（&lt;1分钟）以显示单体物质，并且更长（&gt; 1分钟）以显示高分子量复合物包含ng IF蛋白。如果监测聚集，检查整个凝胶/膜（包括凝胶孔的底部）。 </li><li>运行平行凝胶，并用蛋白质染色剂作为载体对照染色24 。在疾病模型或损伤实验中，总蛋白质染色应用作加载对照，而不是用于"内部"蛋白质（ 例如肌动蛋白，GAPDH）的免疫印迹，因为后者在不同应力条件下发生变化。 </li></ol> 4.组织特异性IF的洗涤剂可溶性和高盐提取物的制备<ol><li>将1mL冰冷的Triton X-100缓冲液加入玻璃管匀浆器中并置于冰上。 </li><li>从液氮储存中取出一小块薄片（约25 mg），并直接放入玻璃均化器中。使用聚四氟乙烯杵匀浆（50冲程），避免产生气泡。始终保持匀浆器和裂解液冷。 </li><li>将裂解液转移至1.5 mL micr离心管在冰上并在预冷却的离心机（4℃）中以20,000×g离心10分钟。 </li><li>将上清液部分收集到单独的管中。这是Triton X-可溶性级分，可用于免疫沉淀（ip）和可洗涤剂溶解的IF蛋白库的分析。注意，步骤4.1-4.4可以重复以实现来自脑组织的更清洁的IF提取物。 </li><li>将1 mL高盐缓冲液加入组织沉淀中，转移至干净的匀浆器中，并行100次。将匀浆转移回微量离心管，并将管置于冷藏室中的旋转振荡器上1小时。 </li><li>在4℃下以20,000×g离心匀浆20分钟。丢弃上清液。 </li><li>将1mL冰冷的PBS / EDTA缓冲液加入到沉淀中，并将其在干净的均化器中匀浆（20次），作为最终的清理步骤*。转移到新管中，在4℃下以20,000×g离心10分钟，得到IF蛋白 -丰富的高盐提取物（HSE）。 *任选地，在该步骤中涡旋而不是均质化。 </li><li>弃去上清液，并将丸粒溶解在300μL已预热的非还原性SDS样品缓冲液中。首先通过移液和涡旋破碎颗粒，然后在95℃下加热样品5分钟。 </li><li>涡流和移液管，以确保颗粒溶解。完全溶解颗粒可能需要几分钟的时间。 </li><li>将所有样品储存在-20°C直到分析。 </li></ol> 5.大容量实验中IF蛋白提取的自动组织裂解<ol><li>对于RNA提取，将裂解缓冲液（每25mg组织600μL缓冲液）置于含有裂解基质D（使用小陶瓷球）的管中，并在组织溶液中脉冲两次25秒。通过以20,000×g离心分离裂解物与基质，并分离进行下一步骤。 </li><li>对于蛋白质提取，放置Trit在X-100（或SDS样品缓冲液，如果准备总裂解物）在裂解管与裂解基质SS（使用单个不锈钢珠）。在测试多个矩阵之后，选择它是因为它产生的IF蛋白质提取物质量与传统的去抖动方法相同。请注意，自动化方法对胰腺和脾脏不是最佳的，标准均质方案应用于这些组织。 </li><li>要进行高盐提取物的制备，使用磁铁从管中取出不锈钢珠，并在4℃下以20,000 xg离心管10分钟。 </li><li>继续执行手册中的步骤4.4（以上）。 </li><li>将所有样品储存在-20°C直到分析。 </li></ol> 6.翻译后修饰的IF蛋白的免疫富集<ol><li>准备PBST缓冲液（0.02％Tween-20在PBS中）。加入10 mL Tween-20至50 mL PBS（pH 7.4）。 </li><li>制备PTM抗体溶液（200μLPBST中的1-10μg抗体）。一般来说，3μg抗体/反应是一个很好的起始条件，如果需要可进一步优化。 </li><li>对于每个反应，将50μL磁珠等分成微量离心管，置于磁体上并吸出珠子储存溶液。 </li><li>通过重新悬浮在抗体溶液中并在室温下孵育20分钟，将珠子与免疫沉淀抗体相结合，并在旋转器（端到端，确保小体积的混合物）上孵育20分钟。 </li><li>将管置于磁铁上并吸出抗体溶液。 </li><li>在200μLPBST中冲洗抗体缀合的珠粒一次，并清洗洗涤缓冲液。 </li><li>将0.6-1mL的组织裂解物加入珠中，通过温和移液混合并在冷室中在旋转器上孵育3小时。 </li><li>将管置于磁体上，除去溶胞产物，并用200μLPBST洗珠5次。最后洗涤步骤后，收集珠子在100 _6; L的PBS（不含Tween-20）并转移到干净的新管中。将管放在磁铁上。 </li><li>吸出PBS并加入100μL非还原样品缓冲液。取出50μL，加入2-ME（5％）以制备还原样品。将样品加热至95°C 5分钟。 </li><li>将ip分数与磁铁上的珠子分开，并将其收集到新的管中。 </li><li>将样品储存在-20°C直到分析。 </li></ol> 7.制备用于质谱分析的IF蛋白质样品<ol><li>在开始研究之前安排与蛋白质组学专家的磋商，因为质谱分析涉及到大量时间和成本。 </li><li>采取特殊预防措施，避免污染。用干净的手套处理所有凝胶，并在干净的容器中孵育，仅使用ddH 2 O（避免使用肥皂）洗涤。 </li><li>根据标准条件，在SDS-PAGE凝胶上运行20-50μL的HSE样品（来自第4和第5部分）。 </l我&gt; <li>用蛋白质污渍染色1小时。漂洗多次，并在ddH 2 O中脱色过夜。去蛋白后，IF蛋白带应易于看到。 </li><li>将凝胶放置在塑料片保护膜之间，扫描并标记将被切除并发送用于分析的条带。 </li><li>使用新的干净剃须刀消除IF蛋白质条带。 </li><li>将凝胶带置于干净的微量离心管中并转移到质谱仪中。 </li></ol>

作者宣称他们没有竞争的经济利益。

使IF蛋白的生物化学表征的方法可用于了解哺乳动物系统中的许多病理生理现象，因为IF蛋白既是细胞和组织应激的标志物和调节剂。目前的方法背后的原理是基于20世纪70年代和80年代开发的初步程序，从细胞和组织中分离，分离和重建IF蛋白，一般采用低盐和高盐溶液和Triton-X100洗涤剂25,30,31,32 ， 33,34,35 。对于支持IF蛋白生化分离的研究的历史见解，请参见最近的评论<su...

<html> IF是一系列蛋白质，在人类中由73个基因编码并分为六种主要类型:I-IV型是细胞质（ 例如上皮和毛发角蛋白（K），肌细胞结蛋白，神经丝，胶质纤维酸性蛋白（GFAP）和别的）; V型是核脂肪;并且VI型是眼镜1中的IF。在其分子组织方面，IF蛋白质具有三个共同的结构域:高度保守的螺旋线圈"棒"结构域，以及球状"头"和"尾"结构域。 IF蛋白质四聚体组装形成短丝前体，其最终并入成熟细丝中，其形成涉及机械保护的动态细胞骨架和核结构结构2 ，应激感测3,4 ，调节转录5和生长以及其他关键细胞功能"&gt; 1,6,7 。 由IF基因错义突变引起的许多人类疾病的存在突出了IF系统的功能重要性，包括神经病，肌病，皮肤脆性障碍，代谢功能障碍和早衰症候群8 。一些IF基因突变不会引起疾病进展的倾向，如肝脏疾病中的简单上皮角蛋白9 。后者是由于上皮中的IF的临界应力保护功能。一般来说，IF在基础条件下是最丰富的细胞蛋白，但在各种类型的应激10中进一步强烈诱导。例如，最近对线虫线虫的蛋白质组范围变化进行的研究表明，多个IF被高度上调并且易于聚集在有机体老化11,12 。由于维持适当的IF结构对于细胞对各种形式的应力10的抵抗是至关重要的，所以IF聚集也可能导致衰老过程中的功能下降。然而，缺乏在不同组织中进行压力检测多种哺乳动物IF蛋白的有机体水平的研究。 IF是高度动态的结构，可以适应蜂窝需求。例如，角蛋白经历可溶性（非丝状）和不溶性（丝状）蛋白质池13之间的生物合成非依赖性循环。在正常的生理条件下，约5％的K8 / K18总库可以在无洗涤剂的缓冲液中提取，相比之下可以溶解在非离子型洗涤剂Nonidet P-40中的约20％，这是与Triton- X100 14 ， </s&gt; 15 。在有丝分裂期间，简单型上皮K8和K1814的溶解度显着增加，这在表皮角蛋白中不太明显，但在波形蛋白和其他III型IF蛋白15,16 中更为明显。 IF蛋白的溶解性质通过磷酸化来进行严格调控，磷酸化是针对灯丝重排和溶解度的关键的翻译后修饰（PTM）17,18,19,20。大多数IF受到保护地点的多个PTM的广泛监管，导致功能变化17 。 这种方法的目的是引入新的IF领域的研究人员进行生物化学提取和多个小鼠组织中IF蛋白研究的分析方法。具体盟军，我们专注于使用高盐提取方法分离IF蛋白，并通过质谱法和PTM靶向抗体评估PTMs的变化。这些方法基于以前发布的程序21，但包括用于提取不同IF蛋白类型的修饰，以揭示通过IF家族调节的常见机制。例如，特定赖氨酸残基处的K8乙酰化调节细丝组织，而超乙酰化促进K8不溶性和聚集体形成22 。最近的全球蛋白质组学分析研究还发现，大多数组织特异性IF蛋白质也是乙酰化的靶标，大多数IF乙酰化位点被限制在高度保守的杆结构域。这突出表明需要适合IF系统的全局分析的方法。我们还介绍了使用Opti自动均质化从多组织分离IF蛋白的快速方法大小溶解基质。所得制剂适用于通过质谱法和其他方法进行下游PTM分析。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Dynabeads Protein G</td>
			<td>ThermoFisher Scientific</td>
			<td>10009</td>
			<td>immunoprecipitation beads</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>ThermoFisher Scientific</td>
			<td>10010049</td>
			<td>for buffers</td>
		</tr>
		<tr>
			<td>Purelink RNA mini kit</td>
			<td>ThermoFisher Scientific</td>
			<td>12183018</td>
			<td>RNA extraction from tissue</td>
		</tr>
		<tr>
			<td>Purelink DNAse set</td>
			<td>ThermoFisher Scientific</td>
			<td>12185010A</td>
			<td>on column DNA digestion</td>
		</tr>
		<tr>
			<td>Dynamag-2</td>
			<td>ThermoFisher Scientific</td>
			<td>12321D</td>
			<td>magnet for use with dynabeads</td>
		</tr>
		<tr>
			<td>GelCode Blue Stain Reagent</td>
			<td>ThermoFisher Scientific</td>
			<td>24592</td>
			<td>mass spectrometry-compatible gel stain</td>
		</tr>
		<tr>
			<td>Pierce ECL Western Blotting Substrate</td>
			<td>ThermoFisher Scientific</td>
			<td>32106</td>
			<td>for use in western blot</td>
		</tr>
		<tr>
			<td>High Capacity cDNA reverse transcription kit</td>
			<td>ThermoFisher Scientific</td>
			<td>4368813</td>
			<td>for use in gene expression analysis</td>
		</tr>
		<tr>
			<td>Proflex 3x32-well PCR System</td>
			<td>ThermoFisher Scientific</td>
			<td>4484073</td>
			<td>PCR system</td>
		</tr>
		<tr>
			<td>PVDF transfer membrane&#160;</td>
			<td>ThermoFisher Scientific</td>
			<td>88520</td>
			<td>for western blot</td>
		</tr>
		<tr>
			<td>Power Up SYBR master mix</td>
			<td>ThermoFisher Scientific</td>
			<td>A25778</td>
			<td>for qPCR analysis</td>
		</tr>
		<tr>
			<td>RNAlater</td>
			<td>ThermoFisher Scientific</td>
			<td>AM7020</td>
			<td>solution for tissue storage prior to RNA isolation</td>
		</tr>
		<tr>
			<td>Novex 4-20% Tris Glycine Gel</td>
			<td>ThermoFisher Scientific</td>
			<td>XP04205BOX</td>
			<td>Precast protein gel</td>
		</tr>
		<tr>
			<td>Anti-Keratin 8 antibody (TS1)</td>
			<td>ThermoFisher Scientific</td>
			<td>MA514428</td>
			<td>for western blot detection of K8</td>
		</tr>
		<tr>
			<td>Anti-Vimentin</td>
			<td>ThermoFisher Scientific</td>
			<td>MA511883</td>
			<td>for western blot detection of vimentin</td>
		</tr>
		<tr>
			<td>Tris Glycine Transfer Buffer (25X)</td>
			<td>ThermoFisher Scientific</td>
			<td>LC3675</td>
			<td>for wet transfer of protein gels</td>
		</tr>
		<tr>
			<td>2X SDS Sample Buffer</td>
			<td>ThermoFisher Scientific</td>
			<td>LC2676</td>
			<td>for preparing protein gel samples</td>
		</tr>
		<tr>
			<td>Tris Glycine SDS Running Buffer (10X)</td>
			<td>ThermoFisher Scientific</td>
			<td>LC26755</td>
			<td>for running protein gels</td>
		</tr>
		<tr>
			<td>Lysing beads - Matrix D</td>
			<td>MP Biomedicals</td>
			<td>116913100</td>
			<td>Lysis beads and matrix tubes for tissue disruption and RNA extraction</td>
		</tr>
		<tr>
			<td>Lysing beads - Matrix SS</td>
			<td>MP Biomedicals</td>
			<td>116921100</td>
			<td>Lysis beads and matrix tubes for tissue disruption and protein extraction</td>
		</tr>
		<tr>
			<td>NanoDrop Lite Spectrophotometer</td>
			<td>ThermoFisher Scientific</td>
			<td>ND-LITE</td>
			<td>measurement of protein and nucleic acid</td>
		</tr>
		<tr>
			<td>Precellys 24 homogenizer</td>
			<td>Bertin Instruments</td>
			<td>EQ03119</td>
			<td>Automated tissue homogenizer</td>
		</tr>
	</tbody>
</table>

isolation of intermediate filament proteins from multiple mouse tissues to study aging-associated post-translational modifications

中间丝（IFs）与肌动蛋白丝和微管一起形成细胞骨架 - 每个细胞的关键结构元件。正常功能的IF为细胞提供机械和应激弹性，而功能失调的IF细胞骨架损害细胞健康，并与许多人类疾病相关。翻译后修饰（PTM）对生理变化和应激条件下的IF动力学进行了严格的调节。因此，监测IFM的PTM签名变化的能力可以有助于在细胞损伤期间作为应激反应者的IF系统的更好的功能理解和最终调节。然而，由超过70个个体基因编码并以组织依赖性方式表达的大量IF蛋白质是排除不同PTM相对重要性的主要挑战。为此，可以监测IF蛋白上的PTMs的方法在生物体层面上，而不是孤立的家庭成员，可以加速这一领域的研究进展。在这里，我们提出生物化学方法，从9种不同的小鼠组织（脑，心脏，肺，肝，小肠，大肠，胰腺，肾脏和）分离IF蛋白的总，洗涤剂可溶性和去污剂抗性分数脾）。我们进一步展示了通过使用裂解基质和不同小鼠组织的自动均质化来快速分离IF蛋白的优化方案。自动化方案对于在具有高样品体积的实验中（例如涉及多个动物和实验组的疾病模型）中的IF进行分析是有用的。所得样品可用于各种下游分析，包括基于质谱的PTM分析。利用这些方法，我们提供新的数据，显示不同小鼠组织（脑和肝）中的IF蛋白在其表达方面发生平行变化老龄化期间的PTMs。

一种使用裂解基质从多个小鼠组织中高度盐提取IF蛋白的新的快速方法。 在这里修饰了分离大部分中间丝蛋白部分与上皮组织的传统方法25,26以包括9种不同的器官和更快速的组织溶解过程。虽然传统方法需要3个手动均质步骤，但修改过程只有1个手动均质步骤，可以?...

Watch this Scientific Journal Video about Isolation of Intermediate Filament Proteins from Multiple Mouse Tissues to Study Aging-associated Post-translational Modifications at JoVE.com

Isolation of Intermediate Filament Proteins from Multiple Mouse Tissues to Study Aging-associated Post-translational Modifications

在这种方法中，我们提出了从多个小鼠组织快速高效地分离中间丝（IF）蛋白质的生物化学方法。分离的IF可用于通过质谱法和其他生化测定法研究翻译后修饰的变化。

从多个小鼠组织分离中间丝状蛋白以研究与老化相关的翻译后修饰

Intermediate filaments (IFs), together with actin filaments and microtubules, form the cytoskeleton &#8211; a critical structural element of every cell. ...

isolation-intermediate-filament-proteins-from-multiple-mouse-tissues

Universidad de Cartagena UDEC

Research

JoVE Journal

Biochemistry

8.4K 次观看.  University of North Carolina at Chapel Hill. 在这种方法中，我们提出了从多个小鼠组织快速高效地分离中间丝（IF）蛋白质的生物化学方法。分离的IF可用于通过质谱法和其他生化测定法研究翻译后修饰的变化。 

视频: Isolation of Intermediate Filament Proteins from Multiple Mouse Tissues to Study Aging-associated Post-translational Modifications

A Method for Measuring RNA N6-methyladenosine Modifications in Cells and Tissues

N6-Methyladenosine (m6A) modifications of RNA are diverse and ubiquitous amongst eukaryotes. They occur in mRNA, rRNA, tRNA, and microRNA. Recent studies have revealed that these reversible RNA modifications affect RNA splicing, translation, degradation, and localization. Multiple physiological processes, like circadian rhythms, stem cell pluripotency, fibrosis, triglyceride metabolism, and obesity are also controlled by m6A modifications. Immunoprecipitation/sequencing, mass spectrometry, and modified northern blotting are some of the methods commonly employed to measure m6A modifications. Herein, we present a northeastern blotting technique for measuring m6A modifications. The current protocol provides good size separation of RNA, better accommodation and standardization for various experimental designs, and clear delineation of m6A modifications in various sources of RNA. While m6A modifications are known to have a crucial impact on human physiology relating to circadian rhythms and obesity, their roles in other (patho)physiological states are unclear. Therefore, investigations on m6A modifications have immense possibility to provide key insights into molecular physiology.

A Method for Measuring RNA N6-methyladenosine Modifications in Cells and Tissues

A modified northern blotting method for measuring N6-methyladenosine (m6A) modifications in RNA is described. The current method can detect modifications in diverse RNAs and controls under various experimental designs.

一种测量方法RNA<em&gt;ñ</em<sup&gt; 6</sup&gt; -methyladenosine细胞和组织修改

N6-Methyladenosine (m6A) modifications of RNA are diverse and ubiquitous amongst eukaryotes. They occur in mRNA, rRNA, tRNA, and microRNA. Recent studies ...

科研

生物化学

Nuclear Magnetic Resonance Spectroscopy for the Identification of Multiple Phosphorylations of Intrinsically Disordered Proteins

Aggregates of the neuronal Tau protein are found inside neurons of Alzheimer's disease patients. Development of the disease is accompanied by increased, abnormal phosphorylation of Tau. In the course of the molecular investigation of Tau functions and dysfunctions in the disease, nuclear magnetic resonance (NMR) spectroscopy is used to identify the multiple phosphorylations of Tau. We present here detailed protocols of recombinant production of Tau in bacteria, with isotopic enrichment for NMR studies. Purification steps that take advantage of Tau's heat stability and high isoelectric point are described. The protocol for in vitro phosphorylation of Tau by recombinant activated ERK2 allows for generating multiple phosphorylations. The protein sample is ready for data acquisition at the issue of these steps. The parameter setup to start recording on the spectrometer is considered next. Finally, the strategy to identify phosphorylation sites of modified Tau, based on NMR data, is explained. The benefit of this methodology compared to other techniques used to identify phosphorylation sites, such as immuno-detection or mass spectrometry (MS), is discussed.

We describe here a method to identify multiple phosphorylations of an intrinsically disordered protein by Nuclear Magnetic Resonance Spectroscopy (NMR), using Tau protein as a case study. Recombinant Tau is isotopically enriched and modified in vitro by a kinase prior to data acquisition and analysis.

核磁共振光谱法本质无序蛋白的磷酸化多元的鉴定

Aggregates of the neuronal Tau protein are found inside neurons of Alzheimer's disease patients. Development of the disease is accompanied by increased, ...

Utilizing a Comprehensive Immunoprecipitation Enrichment System to Identify an Endogenous Post-translational Modification Profile for Target Proteins

It is now well-appreciated that post-translational modifications (PTMs) play an integral role in regulating a protein&#39;s structure and function, which may be essential for a given protein&#39;s role both physiologically and pathologically. Enrichment of PTMs is often necessary when investigating the PTM status of a target protein, because PTMs are often transient and relatively low in abundance. Many pitfalls are encountered when enriching for a PTM of a target protein, such as buffer incompatibility, the target protein antibody is not IP-compatible, loss of PTM signal, and others. The degree of difficulty is magnified when investigating multiple PTMs like acetylation, ubiquitination, SUMOylation 2/3, and tyrosine phosphorylation for a given target protein. Studying a combination of these PTMs may be necessary, as crosstalk between PTMs is prevalent and critical for protein regulation. Often, these PTMs are studied in different lysis buffers and with unique inhibitor compositions. To simplify the process, a unique denaturing lysis system was developed that effectively isolates and preserves these four PTMs; thus, enabling investigation of potential crosstalk in a single lysis system. A unique filter system was engineered to remove contaminating genomic DNA from the lysate, which is a problematic by-product of denaturing buffers. Robust affinity matrices targeting each of the four PTMs were developed in concert with the buffer system to maximize the enrichment and detection of the endogenous states of these four PTMs. This comprehensive PTM detection toolset streamlines the process of obtaining critical information about whether a protein is modified by one or more of these PTMs.

Investigating multiple, endogenous post-translational modifications for a target protein can be extremely challenging. Techniques described here utilize an optimized lysis buffer and filter system developed with specific PTM-targeting, affinity matrices to detect the acetylation, ubiquitination, SUMOylation 2/3, and tyrosine phosphorylation modifications for a target protein using a single, streamlined system.

利用综合沉淀富集系统识别目标蛋白的内源性后向转化剖面

It is now well-appreciated that post-translational modifications (PTMs) play an integral role in regulating a protein&#39;s structure and function, which ...

Leaf Spray Mass Spectrometry: A Rapid Ambient Ionization Technique to Directly Assess Metabolites from Plant Tissues

Plants produce thousands of small molecules that are diverse in their chemical properties. Mass spectrometry (MS) is a powerful technique for analyzing plant metabolites because it provides molecular weights with high sensitivity and specificity. Leaf spray MS is an ambient ionization technique where plant tissue is used for direct chemical analysis via electrospray, eliminating chromatography from the process. This approach to sampling metabolites allows for a wide range of chemical classes to be detected simultaneously from intact plant tissues, minimizing the amount of sample preparation needed. When used with a high-resolution, accurate mass MS, leaf spray MS facilitates the rapid detection of metabolites of interest. It is also possible to collect tandem mass fragmentation data with this technique to facilitate a compound identification. The combination of accurate mass measurements and fragmentation is beneficial in confirming compound identities. The leaf spray MS technique requires only minor modifications to a nanospray ionization source and is a useful tool to further expand the capabilities of a mass spectrometer. Here, fresh leaf tissue from Sceletium tortuosum (Aizoaceae), a traditional medicinal plant from South Africa, is analyzed; numerous mesembrine alkaloids are detected with leaf spray MS.

Leaf spray mass spectrometry is a direct chemical analysis technique that minimizes the sample preparation and eliminates chromatography, allowing for the rapid detection of small molecules from plant tissues.

叶喷雾质谱: 一种快速的环境电离技术直接评价植物组织中的代谢产物

Plants produce thousands of small molecules that are diverse in their chemical properties. Mass spectrometry (MS) is a powerful technique for analyzing ...

Isolation of Glomeruli and In Vivo Labeling of Glomerular Cell Surface Proteins

Proteinuria results from the disruption of the glomerular filter that is composed of the fenestrated endothelium, glomerular basement membrane, and podocytes with their slit diaphragms. The delicate structure of the glomerular filter, especially the slit diaphragm, relies on the interplay of diverse cell surface proteins. Studying these cell surface proteins has so far been limited to in vitro studies or histologic analysis. Here, we present a murine in vivo biotinylation labeling method, which enables the study of glomerular cell surface proteins under physiologic and pathophysiologic conditions. This protocol contains information on how to perfuse mouse kidneys, isolate glomeruli, and perform endogenous immunoprecipitation of a protein of interest. Semi-quantitation of glomerular cell surface abundance is readily available with this novel method, and all proteins accessible to biotin perfusion and immunoprecipitation can be studied. In addition, isolation of glomeruli with or without biotinylation enables further analysis of glomerular RNA and protein as well as primary glomerular cell culture (i.e., primary podocyte cell culture).

Isolation of Glomeruli and In Vivo Labeling of Glomerular Cell Surface Proteins

Here we present a protocol for murine in vivo labeling of glomerular cell surface proteins with biotin. This protocol contains information on how to perfuse mouse kidneys, isolate glomeruli, and perform endogenous immunoprecipitation of the protein of interest.

肾小球的分离及肾小球细胞表面蛋白的活体标记

Proteinuria results from the disruption of the glomerular filter that is composed of the fenestrated endothelium, glomerular basement membrane, and ...

Looking Outwards: Isolation of Cyanobacterial Released Carbohydrate Polymers and Proteins

Cyanobacteria can actively secrete a wide range of biomolecules into the extracellular environment, such as heteropolysaccharides and proteins. The identification and characterization of these biomolecules can improve knowledge about their secretion pathways and help to manipulate them. Furthermore, some of these biomolecules are also interesting in terms of biotechnological applications. Described here are two protocols for easy and rapid isolation of cyanobacterial released carbohydrate polymers and proteins. The method for isolation of released carbohydrate polymers is based on conventional precipitation techniques of polysaccharides in aqueous solutions using organic solvents. This method preserves the characteristics of the polymer and simultaneously avoids the presence of contaminants from cell debris and culture medium. At the end of the process, the lyophilized polymer is ready to be used or characterized or can be subjected to further rounds of purification, depending on the final intended use. Regarding the isolation of the cyanobacterial exoproteome, the technique is based on the concentration of the cell-free medium after removal of the major contaminants by centrifugation and filtration. This strategy allows for reliable isolation of proteins that reach the extracellular milieu via membrane transporters or outer membrane vesicles. These proteins can be subsequently identified using standard mass spectrometry techniques. The protocols presented here can be applied not only to a wide range of cyanobacteria, but also to other bacterial strains. Furthermore, these procedures can be easily tailored according to the final use of the products, purity degree required, and bacterial strain.

Here, protocols for the isolation of cyanobacterial released carbohydrate polymers and isolation of their exoproteomes are described. Both procedures embody key steps to obtain polymers or proteins with high purity degrees that can be used for further analysis or applications. They can also be easily adapted according to specific user needs.

向外观察:分离氰化物释放的碳水化合物聚合物和蛋白质

Cyanobacteria can actively secrete a wide range of biomolecules into the extracellular environment, such as heteropolysaccharides and proteins. The ...

Profiling Ubiquitin and Ubiquitin-like Dependent Post-translational Modifications and Identification of Significant Alterations

Ubiquitin (ub) and ubiquitin-like (ubl) dependent post-translational modifications of proteins play fundamental biological regulatory roles within the cell by controlling protein stability, activity, interactions, and intracellular localization. They enable the cell to respond to signals and to adapt to changes in its environment. Alterations within these mechanisms can lead to severe pathological situations such as neurodegenerative diseases and cancers. The aim of the technique described here is to establish ub/ubls dependent PTMs profiles, rapidly and accurately, from cultured cell lines. The comparison of different profiles obtained from different conditions allows the identification of specific alterations, such as those induced by a treatment for example. Lentiviral mediated cell transduction is performed to create stable cell lines expressing a two-tags (6His and Flag) version of the modifier (ubiquitin or a ubl such as SUMO1 or Nedd8). These tags permit the purification of ubiquitin and therefore of ubiquitinated proteins from the cells. This is done through a two-step purification process: The first one is performed in denaturing conditions using the 6His tag, and the second one in native conditions using the Flag tag. This leads to a highly specific and pure isolation of modified proteins which are subsequently identified and semi-quantified by liquid chromatography followed by tandem mass spectrometry (LC-MS/MS) technology. Easy informatics analysis of MS data using Excel software enables the establishment of PTM profiles by eliminating background signals. These profiles are compared between each condition in order to identify specific alterations which will then be studied more specifically, starting with their validation by standard biochemistry techniques.

This protocol aims at establishing ubiquitin (Ub) and ubiquitin-likes (Ubls) specific proteomes in order to identify alterations of these kind of post-translational modifications (PTMs), associated with a specific condition such as a treatment or a phenotype.

分析泛性基质和泛性基质样的依从性转化后修饰和重大改变的识别

Ubiquitin (ub) and ubiquitin-like (ubl) dependent post-translational modifications of proteins play fundamental biological regulatory roles within the ...

Transverse Sectioning of Mature Rice (Oryza sativa L.) Kernels for Scanning Electron Microscopy Imaging Using Pipette Tips as Immobilization Support

Starch granules (SGs) exhibit different morphologies depending on the plant species, especially in the endosperm of the Poaceae family. Endosperm phenotyping can be used to classify genotypes based on SG morphotype using scanning electron microscopic (SEM) analysis. SGs can be visualized using SEM by slicing through the kernel (pericarp, aleurone layers, and endosperm) and exposing the organellar contents. Current methods require the rice kernel to be embedded in plastic resin and sectioned using a microtome or embedded in a truncated pipette tip and sectioned by hand using a razor blade. The former method requires specialized equipment and is time-consuming, while the latter introduces a new host of problems depending on rice genotype. Chalky rice varieties, particularly, pose a problem for this type of sectioning due to the friable nature of their endosperm tissue. Presented here is a technique for preparing translucent and chalky rice kernel sections for microscopy, requiring only pipette tips and a scalpel blade. Preparing the sections within the confines of a pipette tip prevents rice kernel endosperm from shattering (for translucent or &#8216;vitreous&#8217; phenotypes) and crumbling (for chalky phenotypes). Using this technique, endosperm cell patterning and the structure of intact SGs can be observed.

Transverse Sectioning of Mature Rice Oryza sativa L. Kernels for Scanning Electron Microscopy Imaging Using Pipette Tips as Immobilization Support

This protocol allows for the preparation of transverse sections of cereal seeds (e.g., rice) for the analysis of endosperm and starch granule morphology using scanning electron microscopy.

成熟水稻的横向切片（米 苜蓿L.）使用移液器吸头作为固定支架的扫描电子显微镜成像的内核

Starch granules (SGs) exhibit different morphologies depending on the plant species, especially in the endosperm of the Poaceae family. Endosperm ...

Purification of Tubulin with Controlled Posttranslational Modifications and Isotypes from Limited Sources by Polymerization-Depolymerization Cycles

One important aspect of studies of the microtubule cytoskeleton is the investigation of microtubule behavior in in vitro reconstitution experiments. They allow the analysis of the intrinsic properties of microtubules, such as dynamics, and their interactions with microtubule-associated proteins (MAPs). The &#8220;tubulin code&#8221; is an emerging concept that points to different tubulin isotypes and various posttranslational modifications (PTMs) as regulators of microtubule properties and functions. To explore the molecular mechanisms of the tubulin code, it is crucial to perform in vitro reconstitution experiments using purified tubulin with specific isotypes and PTMs.
To date, this was technically challenging as brain tubulin, which is widely used in in vitro experiments, harbors many PTMs and has a defined isotype composition. Hence, we developed this protocol to purify tubulin from different sources and with different isotype compositions and controlled PTMs, using the classical approach of polymerization and depolymerization cycles. Compared to existing methods based on affinity purification, this approach yields pure, polymerization-competent tubulin, as tubulin resistant to polymerization or depolymerization is discarded during the successive purification steps.
We describe the purification of tubulin from cell lines, grown either in suspension or as adherent cultures, and from single mouse brains. The method first describes the generation of cell mass in both suspension and adherent settings, the lysis step, followed by the successive stages of tubulin purification by polymerization-depolymerization cycles. Our method yields tubulin that can be used in experiments addressing the impact of the tubulin code on the intrinsic properties of microtubules and microtubule interactions with associated proteins.

This protocol describes tubulin purification from small/medium-scale sources such as cultured cells or single mouse brains, using polymerization and depolymerization cycles. The purified tubulin is enriched in specific isotypes or has specific posttranslational modifications and can be used in in vitro reconstitution assays to study microtubule dynamics and interactions.

通过聚合-去聚合周期，通过有限来源的受控后翻译修改和异种型对图布林进行净化

One important aspect of studies of the microtubule cytoskeleton is the investigation of microtubule behavior in in vitro reconstitution experiments. They ...

Purification of Ubiquitinated p53 Proteins from Mammalian Cells

Ubiquitination is a type of posttranslational modification that regulates not only the stability but also the localization and function of a substrate protein. The ubiquitination process occurs intracellularly in eukaryotes and regulates almost all basic cellular biological processes. Purification of ubiquitinated proteins aids the investigation of the role of ubiquitination in controlling the function of substrate proteins. Here, a step-by-step procedure to purify ubiquitinated proteins in mammalian cells is described with the p53 tumor suppressor protein as an example. Ubiquitinated p53 proteins were purified under stringent nondenaturing and denaturing conditions. Total cellular Flag-tagged p53 protein was purified with anti-Flag antibody-conjugated agarose under nondenaturing conditions. Alternatively, total cellular His-tagged ubiquitinated protein was purified using nickel-charged resin under denaturing conditions. Ubiquitinated p53 proteins in the eluates were successfully detected with specific antibodies. Using this procedure, the ubiquitinated forms of a given protein can be efficiently purified from mammalian cells, facilitating studies on the roles of ubiquitination in regulating protein function.

The protocol describes a step-by-step method to purify ubiquitinated proteins from mammalian cells using the p53 tumor suppressor protein as an example. Ubiquitinated p53 proteins were purified from cells under stringent nondenaturing and denaturing conditions.

从哺乳动物细胞中纯化泛素化p53蛋白

Ubiquitination is a type of posttranslational modification that regulates not only the stability but also the localization and function of a substrate ...

从多个小鼠组织分离中间丝状蛋白以研究与老化相关的翻译后修饰

摘要

探索更多视频

此视频中的章节

一种测量方法RNA

核磁共振光谱法本质无序蛋白的磷酸化多元的鉴定

利用综合沉淀富集系统识别目标蛋白的内源性后向转化剖面

叶喷雾质谱: 一种快速的环境电离技术直接评价植物组织中的代谢产物

肾小球的分离及肾小球细胞表面蛋白的活体标记

向外观察:分离氰化物释放的碳水化合物聚合物和蛋白质

分析泛性基质和泛性基质样的依从性转化后修饰和重大改变的识别

成熟水稻的横向切片（米苜蓿L.）使用移液器吸头作为固定支架的扫描电子显微镜成像的内核

通过聚合-去聚合周期，通过有限来源的受控后翻译修改和异种型对图布林进行净化

从哺乳动物细胞中纯化泛素化p53蛋白

从多个小鼠组织分离中间丝状蛋白以研究与老化相关的翻译后修饰

摘要

探索更多视频

此视频中的章节

一种测量方法RNA

核磁共振光谱法本质无序蛋白的磷酸化多元的鉴定

利用综合沉淀富集系统识别目标蛋白的内源性后向转化剖面

叶喷雾质谱: 一种快速的环境电离技术直接评价植物组织中的代谢产物

肾小球的分离及肾小球细胞表面蛋白的活体标记

向外观察:分离氰化物释放的碳水化合物聚合物和蛋白质

分析泛性基质和泛性基质样的依从性转化后修饰和重大改变的识别

成熟水稻的横向切片（米 苜蓿L.）使用移液器吸头作为固定支架的扫描电子显微镜成像的内核

通过聚合-去聚合周期，通过有限来源的受控后翻译修改和异种型对图布林进行净化

从哺乳动物细胞中纯化泛素化p53蛋白

成熟水稻的横向切片（米苜蓿L.）使用移液器吸头作为固定支架的扫描电子显微镜成像的内核