This work was supported by Inserm. Marion Bouvet is a fellow of the French Ministry for Research and Technology. Annie Turkieh is a fellow granted by European Union FP7 HOMAGE (305507).

该协议符合我们的机构人类研究伦理委员会的指导方针。从地区输血中心（里尔，法国）获得从健康人供体棕黄色大衣。从淡黄色大衣得到的样品被声明为一个INSERM集合（N°DC2010-1209）。 1.材料和文化传媒准备<ol><li>稀释10倍的磷酸盐缓冲盐水（PBS），在无菌蒸馏水中，得到的1×PBS。 </li><li>作的RPMI 1640培养基补充有庆大霉素（40微克/毫升）和L-谷氨酰胺（2毫摩尔），有和没有10％合并的人血清。 </li></ol>单核细胞 - 巨噬2.原代培养（MDM） <ol><li>稀buffycoat（25毫升）用1×PBS。在Ficoll / leucosep管和离心谨慎地装载在1600×g离心20分钟，在室温下进行。 </li><li>收集单核细胞的界面处进新管中。洗涤3次，用10ml。一次含0.1％乙二胺四PBS中乙酸（EDTA）的通过连续离心以1000×g离心，370×g离心和160×g离心，每次10分钟，然后再一次在1×PBS中单独以160×g离心10分钟。 </li><li>重新悬浮于5ml RPMI-1640培养基将细胞沉淀无血清和种子细胞35毫米培养皿中，每皿1×10 6个细胞的密度。 </li><li>沉降90分钟，在孵化后，弃去含有非贴壁细胞上清。洗涤贴壁细胞，包括单核细胞，3倍，用1ml的PBS;然后加入1毫升含有10％的新鲜培养基（体积/体积）人血清与先前的无血清细胞。 </li><li>培养6天后的，以产生替代性分化的巨噬细胞（M2）中，添加重组人IL-4（15毫微克/毫升），保持6天。然后，治疗分化的巨噬细胞用脂多糖（100毫微克/毫升）在12天4小时，得到M1巨噬细胞。 </li></ol> 3.提取M1和M2巨噬细胞蛋白的二维电泳<ol><li>洗macroph年龄三次用25mM的Tris，pH 7.4中和废料中含有30毫摩尔Tris pH为8,4％3缓冲器 - [（3-胆酰胺丙基）二甲氨基] -1-丙磺酸盐（CHAPS），2M硫脲和7M尿素。 </li><li>使用合适的1.5 ml微量离心管5分钟，在冰中并储存在-20℃的混合器裂解细胞。 </li><li>使用市售的Bradford试剂测定蛋白质浓度。存储该蛋白质的100μl的等分试样在-20℃下直到使用。 </li></ol> 4.等电聚焦注:请在黑暗中的所有标记的​​程序。 <ol><li>减少5微克每一个样品（M1和M2巨噬细胞），2毫摩尔Tris（2-羧乙基）膦（TCEP）处理1小时，调整为9微升用裂解缓冲液中，在37℃。 </li><li>添加菁3（Cy3标记）与M1提取物和M2的巯基反应性染料提取物为0.8纳米/微克蛋白的浓度。添加花青-5（Cy5标记），以M1和M2中提取在0.8纳米/微克蛋白的浓度和孵育为30分钟，在37℃。 </li><li>停止通过加入含7M尿素，2M硫脲，4％CHAPS，130毫摩尔二硫苏糖醇（DTT）和2％pharmalytes样品缓冲液等体积​​的反应。 </li><li>混合Cy3标记M1的样品用Cy5标记M2的样品中的单手和Cy3标记M2的样本与另一个手Cy5标记M1的样品。 </li><li>再水化的固相pH梯度（IPG）条（240毫米，pH值3-10的线性梯度）用450微升标记的混合样品在含7M尿素，在等电2M硫脲，4％CHAPS缓冲聚焦（IEF）电池系统为24小时，不施加任何电流。 </li><li>执行，在300伏（V）聚焦3小时，然后在梯度1,000V，6小时，在梯度的8000 V 3小时，最后在8000 V 3小时。 </li></ol> 5.第二维注:请在黑暗中所有的电泳程序。 <ol><li>孵育IPG胶条的平衡缓冲液含有荷兰国际集团为0.1mM的Tris-HCl（pH8）中，6M尿素，2％（重量/体积）十二烷基硫酸钠（SDS）和30％（体积/体积）甘油10分钟。 </li><li>传输平衡的IPG胶条用于第二维（SDS-聚丙烯酰胺凝胶电泳（PAGE））到12.5％PAGE凝胶上，并密封与低熔点琼脂糖。 </li><li>进行电泳，在20℃下使用的Ettan-Daltsix系统，在70伏的恒定电压过夜，然后300伏，直到溴酚蓝前沿到达凝胶的底部。 </li></ol> 6.图像采集和生物信息学分析<ol><li>扫描凝胶投两低荧光玻璃板之间具有DIGE成像扫描器在五百八分之五百三十二处为Cy3和六百七十○分之六百三十三激发波长/发射波长为Cy5标记，以具有100微米的像素尺寸得到的图像。 </li><li>用商业软件进行图像分析如先前详述12。 </li><li>计算和规范点卷中的每个图像。分配一个标准化的现货量为道具检测凝胶中的每个点的总价值的钮用来。 </li><li>通过比较这两个基团（M1和M2）之间的归一化点体积值分析对于每种类型的巨噬细胞中的蛋白斑点体积的差异。考虑光斑的卷之间的差异是显著如果变化为1.5倍（p &lt;0.05，单向ANOVA分析）。 </li></ol>

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There are no declared conflict of interest.

本文所描述的协议详细说明了分析巨噬细胞中，M1（促炎）和M2（抗炎）的两个亚型的各种刺激的影响的方法。的M1和M2巨噬细胞的原代培养物是从单核细胞分化得到的先前公布的7。 的2D DIGE凝胶电泳的方法需要特殊的材料和设备，如等电聚焦，以扫描在对Cy3和Cy5，一个扫描器，用于荧光激发/发射波长下的凝胶两次细胞对于等电聚焦，低荧光板用于SDS-PAGE上和2D软件。这...

巨噬细胞是异质的，塑料的细胞，能够获得不同的功能表型。 在体内，这些细胞对大量的各种微环境signalssuch作为微生物产物，细胞因子， 等等 。1的响应。 在体外 ，所述促炎性表型（M1）的巨噬细胞可通过脂多糖（LPS）和抗炎表型（M2），通过某些细胞因子，如白介素-4（IL-4）诱导。此外，巨噬细胞可以从一个激活的M1至M2的表型转换，而相反地，在特定的信号2。 根据不同的表型，巨噬细胞将具有不同的功能。 M1巨噬细胞产生促炎细胞因子，如肿瘤坏死因子α（TNF-α），杀灭微生物或肿瘤细胞的3个细胞。与此相反，M2巨噬细胞阻止这些炎性应答状在伤口愈合和纤维化由产生抗inflammatorÝ因子如TGF-β3,4。 从健康供体的人外周血单核细胞通过Ficoll密度梯度离心法如使用适于从Boyum 6的技术先前描述5中分离得到。在培养的巨噬细胞后6天的原代培养7可以分化成M1或M2的表型。 蛋白质表达或根据各种受控刺激，如宿主的致病性或微生物毒素的巨噬细胞的两种亚型之间的蛋白质变化的分析，将是有益的破译亲和抗炎的巨噬细胞的功能。 蛋白质组学是用于直接监控的蛋白质，是具体地上调或下调的下各种刺激培养的人体巨噬细胞的独特工具。荧光染料已解决了某些二维凝胶电泳的局限性，如低灵敏度和图像分析8 &lt;<html> / SUP&gt;。染料与半胱氨酸残基反应而增加的检测灵敏度相比，那些与赖氨酸残基9的反应。在先前的研究中，我们证明了DIGE饱和标记的有用稀少样品10的分析结果相比较，以经典的银染的二维电泳11。这种技术是在快速分析巨噬细胞的两种亚型之间或从同一亚型未处理和处理的巨噬细胞之间的蛋白质修饰很有帮助。 此蛋白质组技术的优点是具有由2D凝胶12分析获得的蛋白质的大小和翻译后修饰的信息。应当考虑到，这不是一个高通量技术，限制了可被分析的样品的数目。基于质谱的高通量测定法评论了发展最近13可以改善这一点。 _content"&gt;这里，我们提出了如何通过电泳，等电聚焦的过程中执行从培养的巨噬细胞的蛋白提取2D DIGE分析和SDS-PAGE，以及对适当的2D软件的有用信息。

<table><tbody><tr><td>RPMI 1640</td><td>Invitrogen</td><td>31870-074</td><td></td></tr><tr><td>PBX 10 X</td><td>Invitrogen</td><td>14200-083</td><td></td></tr><tr><td>L-glutamine-200 mM-100X</td><td>Invitrogen</td><td>25030-024</td><td></td></tr><tr><td>gentamycin 10 mg/ml</td><td>Invitrogen</td><td>15710-049</td><td></td></tr><tr><td>human serum</td><td>Invitrogen</td><td>34005100</td><td></td></tr><tr><td>Ficoll d = 1,077</td><td>ATGC</td><td>L6115</td><td></td></tr><tr><td>Leucosep</td><td>Dutscher</td><td>16760</td><td></td></tr><tr><td>&amp;nbsp;6-wells plate PRIMARIA&amp;nbsp;</td><td>Becton Dickinson</td><td>353846</td><td></td></tr><tr><td>IL-4</td><td>Promocell</td><td>B-61410</td><td></td></tr><tr><td>lipopolysaccharide</td><td>Sigma-Aldrich</td><td>L-2654</td><td></td></tr><tr><td>Giemsa</td><td>Fluka</td><td>48900</td><td></td></tr><tr><td>EDTA MM372,2</td><td>Research Organics&amp;nbsp;</td><td>3.00E+01</td><td></td></tr><tr><td>Filter 0.22 &amp;micro;m</td><td>Millipore</td><td>SCGPTORE</td><td></td></tr><tr><td>100 ml cylinder</td><td>Corning</td><td>430182</td><td></td></tr><tr><td>TCEP</td><td>Interchim</td><td>UP242214</td><td></td></tr><tr><td>Bradford reagent</td><td>Bio-Rad</td><td>5000006</td><td></td></tr><tr><td>Cy3+Cy5-reactive dye</td><td>GE Healthcare</td><td>25-8009-83</td><td></td></tr><tr><td>IPG strip 3-10 24cm</td><td>GE Healthcare</td><td>17-6002-44</td><td></td></tr><tr><td>Protean IEF cell</td><td>Bio-Rad</td><td>165-4000</td><td></td></tr><tr><td>Low-melting agarose</td><td>Invitrogen</td><td>15517-014</td><td></td></tr><tr><td>Ettan-Daltsix system</td><td>GE Healthcare</td><td>80-6485-08</td><td></td></tr><tr><td>Ettan DIGE Imager scanner</td><td>GE Healthcare</td><td></td><td></td></tr><tr><td>Progenesis Samespot</td><td>Non linear dynamics</td><td></td><td></td></tr><tr><td>50 ml tubes</td><td>any supplier</td><td>n/a</td><td></td></tr><tr><td>15 ml tubes</td><td>any supplier</td><td>n/a</td><td></td></tr><tr><td>CHAPS</td><td>Sigma-Aldrich</td><td>C5070</td><td></td></tr><tr><td>Ur&amp;eacute;e</td><td>Merk</td><td>108484-500</td><td></td></tr><tr><td>Thiour&amp;eacute;e</td><td>Sigma-Aldrich</td><td>T7875</td><td></td></tr><tr><td>DTT</td><td>Bio-Rad</td><td>1610611</td><td></td></tr><tr><td>APS</td><td>Sigma-Aldrich</td><td>A3678</td><td></td></tr><tr><td>TEMED</td><td>Sigma-Aldrich</td><td>T9281</td><td></td></tr><tr><td>Tris Base</td><td>Sigma-Aldrich</td><td>T1503</td><td></td></tr><tr><td>Tris HCl</td><td>Sigma-Aldrich</td><td>T3253</td><td></td></tr><tr><td>Pharmalytes 3-10</td><td>GE Healthcare</td><td>17-0456-01</td><td></td></tr><tr><td>SDS</td><td>Sigma-Aldrich</td><td>L3773</td><td></td></tr><tr><td>Bromophenol blue</td><td>Sigma-Aldrich</td><td>114391</td><td></td></tr><tr><td>Glycerol</td><td>Sigma-Aldrich</td><td>G6279</td><td></td></tr><tr><td>Acrylamide 40%</td><td>Bio-Rad</td><td>161-0148</td><td></td></tr><tr><td>2D clean Up</td><td>GE Healthcare</td><td>80-6454-51</td><td></td></tr><tr><td>Glycine</td><td>Sigma-Aldrich</td><td>G7126</td><td></td></tr><tr><td>Dim&amp;eacute;thylformamide</td><td>Sigma-Aldrich</td><td>22705-6</td><td></td></tr><tr><td>electrode wicks</td><td>Bio-Rad</td><td>165-4071</td><td></td></tr></tbody></table>

proteomic profiling of macrophages by 2d electrophoresis

The goal of the two-dimensional (2D) electrophoresis protocol described here is to show how to analyse the phenotype of human cultured macrophages. The key role of macrophages has been shown in various pathological disorders such as inflammatory, immunological, and infectious diseases. In this protocol, we use primary cultures of human monocyte-derived macrophages that can be differentiated into the M1 (pro-inflammatory) or the M2 (anti-inflammatory) phenotype. This in vitro model is reliable for studying the biological activities of M1 and M2 macrophages and also for a proteomic approach. Proteomic techniques are useful for comparing the phenotype and behaviour of M1 and M2 macrophages during host pathogenicity. 2D gel electrophoresis is a powerful proteomic technique for mapping large numbers of proteins or polypeptides simultaneously. We describe the protocol of 2D electrophoresis using fluorescent dyes, named 2D Differential Gel Electrophoresis (DIGE). The M1 and M2 macrophages proteins are labelled with cyanine dyes before separation by isoelectric focusing, according to their isoelectric point in the first dimension, and their molecular mass, in the second dimension. Separated protein or polypeptidic spots are then used to detect differences in protein or polypeptide expression levels. The proteomic approaches described here allows the investigation of the macrophage protein changes associated with various disorders like host pathogenicity or microbial toxins.

执行适当的差动蛋白质组学分析，待分析样品的处理应加以核实。 在给出的例子中，需要的巨噬细胞的细胞培养物的质量为形态学和分子方面的先前公布的5。单核细胞分化成巨噬细胞和培养物的均一性，随后通过相显微镜。 图1显示的M1和M2巨噬细胞的原代培养物的一个例子。如图所示，我们证实M1（ 图1A，B）和M 2（ 图1C，D） ​​的</s...

Watch this Scientific Journal Video about Proteomic Profiling of Macrophages by 2D Electrophoresis at JoVE.com

Proteomic Profiling of Macrophages by 2D Electrophoresis

Macrophages are the key cells involved in host pathogenicity. Macrophages display phenotypic and functional diversity that can be analysed and detected by proteomic analysis. This article describes how to perform 2D electrophoresis of primary cultures of human macrophages differentiated into M1 or M2 phenotype.

巨噬细胞通过二维电泳蛋白组学分析

The goal of the two-dimensional (2D) electrophoresis protocol described here is to show how to analyse the phenotype of human cultured macrophages. The ...

proteomic-profiling-of-macrophages-by-2d-electrophoresis

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

12.6K 次观看.  University Lille Nord de France. Macrophages are the key cells involved in host pathogenicity. Macrophages display phenotypic and functional diversity that can be analysed and detected by proteomic analysis. This article describes how to perform 2D electrophoresis of primary cultures of human macrophages differentiated into M1 or M2 phenotype.

视频: Proteomic Profiling of Macrophages by 2D Electrophoresis

Two-dimensional Gel Electrophoresis Coupled with Mass Spectrometry Methods for an Analysis of Human Pituitary Adenoma Tissue Proteome

Human pituitary adenoma (PA) is a common tumor that occurs in the human pituitary gland in the hypothalamus-pituitary-targeted organ axis systems, and may be classified as either clinically functional or nonfunctional PA (FPA and NFPA). NFPA is difficult for early stage diagnosis and therapy due to barely elevating hormones in the blood compared to FPA. Our long-term goal is to use proteomics methods to discover reliable biomarkers for clarification of PA molecular mechanisms and recognition of effective diagnostic, prognostic markers and therapeutic targets. Effective two-dimensional gel electrophoresis (2DE) coupled with mass spectrometry (MS) methods were presented here to analyze human PA proteomes, including preparation of samples, 2D gel electrophoresis, protein visualization, image analysis, in-gel trypsin digestion, peptide mass fingerprint (PMF), and tandem mass spectrometry (MS/MS). 2-Dimensional gel electrophoresis matrix-assisted laser desorption/ionization mass spectrometry PMF (2DE-MALDI MS PMF), 2DE-MALDI MS/MS, and 2DE-liquid chromatography (LC) MS/MS procedures have been successfully applied in an analysis of NFPA proteome. With the use of a high-sensitivity mass spectrometer, many proteins were identified with the 2DE-LC-MS/MS method in each 2D gel spot in an analysis of complex PA tissue to maximize the coverage of human PA proteome.

Here, we present a two-dimensional gel electrophoresis (2DE) coupled with mass spectrometry (MS) to separate and identify human pituitary adenoma tissue proteome, which presents a good and reproducible 2DE pattern. Many proteins are observed in each 2DE spot when analyzing complex cancer proteome with the use of high-sensitivity MS.

二维凝胶电泳结合质谱法分析人垂体腺瘤组织蛋白质组

Human pituitary adenoma (PA) is a common tumor that occurs in the human pituitary gland in the hypothalamus-pituitary-targeted organ axis systems, and may ...

科研

癌症研究

Selective Labelling of Cell-surface Proteins using CyDye DIGE Fluor Minimal Dyes

Surface  proteins are central to the cell's ability to react to its environment and to interact with neighboring cells. They are known to be inducers of almost all intracellular signaling. Moreover, they play an important role in environmental adaptation and drug treatment, and are often involved in disease pathogenesis and pathology (1). Protein-protein interactions are intrinsic to signaling pathways, and to gain more insight in these complex biological processes, sensitive and reliable methods are needed for studying cell surface proteins. Two-dimensional (2-D) electrophoresis is used extensively for detection of biomarkers and other targets in complex protein samples to study differential changes. Cell surface proteins, partly due to their low abundance (1 2% of cellular proteins), are difficult to detect in a 2-D gel without fractionation or some other type of enrichment. They are also often poorly represented in 2-D gels due to their hydrophobic nature and high molecular weight (2). In this study, we present a new protocol for intact cells using CyDye  DIGE Fluor minimal dyes for specific labeling and detection of this important group of proteins. The results showed specific labeling of a large number of cell surface proteins with minimal labeling of intracellular proteins. This protocol is rapid, simple to use, and all three CyDye DIGE Fluor minimal dyes (Cy 2, Cy 3 and Cy 5) can be used to label cell-surface proteins. These features allow for multiplexing using the 2-D Fluorescence Difference Gel Electrophoresis (2-D DIGE) with Ettan  DIGE technology and analysis of protein expression changes using DeCyder  2-D Differential Analysis Software. The level of cell-surface proteins was followed during serum starvation of CHO cells for various lengths of time (see Table 1). Small changes in abundance were detected with high accuracy, and results are supported by defined statistical methods.

A simple and specific method was demonstrated for fluorescent labeling and enhanced detection of cell surface proteins without a fractionation step. Differential abundance in cell surface proteins was analyzed using two-dimensional (2-D) electrophoresis and Ettan&#x2122; DIGE technology.

使用CyDye DIGE Fluor公司最小染料的细胞表面蛋白的选择性标签

Surface  proteins are central to the cell's ability to react to its environment and to interact with neighboring cells. They are known to be inducers of ...

生物学

Proteomic Analysis of Human Macrophage Polarization Under a Low Oxygen Environment

Macrophages are innate immune cells involved in a number of physiological functions ranging from responses to infectious pathogens to tissue homeostasis. The various functions of these cells are related to their activation states, which is also called polarization. The precise molecular description of these various polarizations is a priority in the field of macrophage biology. It is currently acknowledged that a multidimensional approach is necessary to describe how polarization is controlled by environmental signals. In this report, we describe a protocol designed to obtain the proteomic signature of various polarizations in human macrophages. This protocol is based on a label-free quantification of macrophage protein expression obtained from in-gel fractionated and Lys C/trypsin-digested cellular lysis content. We also provide a protocol based on in-solution digestion and isoelectric focusing fractionation to use as an alternative. Because oxygen concentration is a relevant environmental parameter in tissues, we use this protocol to explore how atmospheric composition or a low oxygen environment affects the classification of macrophage polarization.

We present a protocol to obtain proteomic signatures of human macrophages and apply this to determination of the impact of a low oxygen environment on macrophage polarization.

低氧环境下人巨噬细胞极化的蛋白质组学分析

Macrophages are innate immune cells involved in a number of physiological functions ranging from responses to infectious pathogens to tissue homeostasis. ...

生物化学

Whole-cell MALDI-TOF Mass Spectrometry is an Accurate and Rapid Method to Analyze Different Modes of Macrophage Activation

MALDI-TOF is an extensively used mass spectrometry technique in chemistry and biochemistry. It has been also applied in medicine to identify molecules and biomarkers. Recently, it has been used in microbiology for the routine identification of bacteria grown from clinical samples, without preparation or fractionation steps. We and others have applied this whole-cell MALDI-TOF mass spectrometry technique successfully to eukaryotic cells. Current applications range from cell type identification to quality control assessment of cell culture and diagnostic applications. Here, we describe its use to explore the various polarization phenotypes of macrophages in response to cytokines or heat-killed bacteria. It allowed the identification of macrophage-specific fingerprints that are representative of the diversity of proteomic responses of macrophages. This application illustrates the accuracy and simplicity of the method. The protocol we described here may be useful for studying the immune host response in pathological conditions or may be extended to wider diagnostic applications.

This protocol describes the use of whole-cell MALDI-TOF mass spectrometry on eukaryotic cells. Here, we illustrate the accuracy of this technique by analyzing the multiple activation states of macrophages in response to their microenvironment.

全细胞MALDI-TOF质谱法是一种准确，快速的方法来分析巨噬细胞活化的不同模式

MALDI-TOF is an extensively used mass spectrometry technique in chemistry and biochemistry. It has been also applied in medicine to identify molecules and ...

免疫与感染

Consensus Brain-derived Protein, Extraction Protocol for the Study of Human and Murine Brain Proteome Using Both 2D-DIGE and Mini 2DE Immunoblotting

Two-dimensional gel electrophoresis (2DE) is a powerful tool to uncover proteome modifications potentially related to different physiological or pathological conditions. Basically, this technique is based on the separation of proteins according to their isoelectric point in a first step, and secondly according to their molecular weights by SDS polyacrylamide gel electrophoresis (SDS-PAGE). In this report an optimized sample preparation protocol for little amount of human post-mortem and mouse brain tissue is described. This method enables to perform both two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) and mini 2DE immunoblotting. The combination of these approaches allows one to not only find new proteins and/or protein modifications in their expression thanks to its compatibility with mass spectrometry detection, but also a new insight into markers validation. Thus, mini-2DE coupled to western blotting permits to identify and validate post-translational modifications, proteins catabolism and provides a qualitative comparison among different conditions and/or treatments. Herein, we provide a method to study components of protein aggregates found in AD and Lewy body dementia such as the amyloid-beta peptide and the alpha-synuclein. Our method can thus be adapted for the analysis of the proteome and insoluble proteins extract from human brain tissue and mice models too. In parallel, it may provide useful information for the study of molecular and cellular pathways involved in neurodegenerative diseases as well as potential novel biomarkers and therapeutic targets.

A common protein extraction protocol using urea/thiourea/SDS buffer for human and mice brain tissue allows indentification of proteins by 2D-DIGE and their subsequent characterization by mini 2DE immunoblotting. This method enables one to obtain more reproducible and reliable results from human biopsies and experimental models.

共识脑源性蛋白，人类和小鼠脑蛋白质组使用两个2D-DIGE和Mini双向电泳免疫印迹法研究提取协议

Two-dimensional gel electrophoresis (2DE) is a powerful tool to uncover proteome modifications potentially related to different physiological or ...

神经科学

From a 2DE-Gel Spot to Protein Function: Lesson Learned From HS1 in Chronic Lymphocytic Leukemia

The identification of molecules involved in tumor initiation and progression is fundamental for understanding disease&#8217;s biology and, as a consequence, for the clinical management of patients. In the present work we will describe an optimized proteomic approach for the identification of molecules involved in the progression of Chronic Lymphocytic Leukemia (CLL). In detail, leukemic cell lysates are resolved by 2-dimensional Electrophoresis (2DE) and visualized as &#8220;spots&#8221; on the 2DE gels. Comparative analysis of proteomic maps allows the identification of differentially expressed proteins (in terms of abundance and post-translational modifications) that are picked, isolated and identified by Mass Spectrometry (MS). The biological function of the identified candidates can be tested by different assays (i.e. migration, adhesion and F-actin polymerization), that we have optimized for primary leukemic cells.

Here we describe a protocol that couples two proteomic techniques, namely 2-dimensional Electrophoresis (2DE) and Mass Spectrometry (MS), to identify differentially expressed/post-translational modified proteins among two or more groups of primary samples. This approach, together with functional experiments, allows the identification and characterization of prognostic markers/therapeutic targets.

从2DE凝胶点在蛋白质功能:在课慢性淋巴细胞白血病学到HS1

The identification of molecules involved in tumor initiation and progression is fundamental for understanding disease&#8217;s biology and, as a ...

医学

Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE) for Analysis of Multiprotein Complexes from Cellular Lysates

Multiprotein complexes (MPCs) play a crucial role in cell signalling, since most proteins can be found in functional or regulatory complexes with other proteins (Sali, Glaeser et al. 2003). Thus, the study of protein-protein interaction networks requires the detailed characterization of MPCs to gain an integrative understanding of protein function and regulation. For identification and analysis, MPCs must be separated under native conditions. In this video, we describe the analysis of MPCs by blue native polyacrylamide gel electrophoresis (BN-PAGE). BN-PAGE is a technique that allows separation of MPCs in a native conformation with a higher resolution than offered by gel filtration or sucrose density ultracentrifugation, and is therefore useful to determine MPC size, composition, and relative abundance (Sch&auml;gger and von Jagow 1991); (Sch&auml;gger, Cramer et al. 1994). By this method, proteins are separated according to their hydrodynamic size and shape in a polyacrylamide matrix. Here, we demonstrate the analysis of MPCs of total cellular lysates, pointing out that lysate dialysis is the crucial step to make BN-PAGE applicable to these biological samples. Using a combination of first dimension BN- and second dimension SDS-PAGE, we show that MPCs separated by BN-PAGE can be further subdivided into their individual constituents by SDS-PAGE. Visualization of the MPC components upon gel separation is performed by standard immunoblotting. As an example for MPC analysis by BN-PAGE, we chose the well-characterized eukaryotic 19S, 20S, and 26S proteasomes.

Blue Native Polyacrylamide Gel Electrophoresis BN-PAGE for Analysis of Multiprotein Complexes from Cellular Lysates

In this video, we describe the characterization of multiprotein complexes (MPCs) by blue native polyacrylamide gel electrophoresis (BN-PAGE). In a first dimension, dialyzed cellular lysates are separated by BN-PAGE to identify individual MPCs. In a second dimension SDS-PAGE, MPCs of interest are further subdivided to analyze their constituents by immunoblotting.

蓝原生聚丙烯酰胺凝胶电泳（BN - PAGE）分析，从细胞裂解液多蛋白复合物

Multiprotein complexes (MPCs) play a crucial role in cell signalling, since most proteins can be found in functional or regulatory complexes with other ...

Isolation, Characterization, and Purification of Macrophages from Tissues Affected by Obesity-related Inflammation

Obesity promotes a chronic inflammatory state that is largely mediated by tissue-resident macrophages as well as monocyte-derived macrophages. Diet-induced obesity (DIO) is a valuable model in studying the role of macrophage heterogeneity; however, adequate macrophage isolations are difficult to acquire from inflamed tissues. In this protocol, we outline the isolation steps and necessary troubleshooting guidelines derived from our studies for obtaining a suitable population of tissue-resident macrophages from mice following 18 weeks of high-fat (HFD) or high-fat/high-cholesterol (HFHCD) diet intervention. This protocol focuses on three hallmark tissues studied in obesity and atherosclerosis including the liver, white adipose tissues (WAT), and the aorta. We highlight how dualistic usage of flow cytometry can achieve a new dimension of isolation and characterization of tissue-resident macrophages. A fundamental section of this protocol addresses the intricacies underlying tissue-specific enzymatic digestions and macrophage isolation, and subsequent cell-surface antibody staining for flow cytometric analysis. This protocol addresses existing complexities underlying fluorescent-activated cell sorting (FACS) and presents clarifications to these complexities so as to obtain broad range characterization from adequately sorted cell populations. Alternate enrichment methods are included for sorting cells, such as the dense liver, allowing for flexibility and time management when working with FACS. In brief, this protocol aids the researcher to evaluate macrophage heterogeneity from a multitude of inflamed tissues in a given study and provides insightful troubleshooting tips that have been successful for favorable cellular isolation and characterization of immune cells in DIO-mediated inflammation.

This protocol allows a researcher to isolate and characterize tissue-resident macrophages in various hallmark inflamed tissues extracted from diet-induced models of metabolic disorders.

受肥胖相关炎症组织的巨噬细胞的分离、鉴定和纯化

Obesity promotes a chronic inflammatory state that is largely mediated by tissue-resident macrophages as well as monocyte-derived macrophages. ...

Label-Free Quantitative Proteomics Workflow for Discovery-Driven Host-Pathogen Interactions

The technological achievements of mass spectrometry (MS)-based quantitative proteomics opens many undiscovered avenues for analyzing an organism&#8217;s global proteome under varying conditions. This powerful strategy applied to the interactions of microbial pathogens with the desired host comprehensively characterizes both perspectives towards infection. Herein, the workflow describes label-free quantification (LFQ) of the infectome of Cryptococcus neoformans, a fungal facultative intracellular pathogen that is the causative agent of the deadly disease cryptococcosis, in the presence of immortalized macrophage cells. The protocol details the proper protein preparation techniques for both pathogen and mammalian cells within a single experiment, resulting in appropriate peptide submission for liquid-chromatography (LC)-MS/MS analysis. The high throughput generic nature of LFQ allows a wide dynamic range of protein identification and quantification, as well as transferability to any host-pathogen infection setting, maintaining extreme sensitivity. The method is optimized to catalogue extensive, unbiased protein abundance profiles of a pathogen within infection-mimicking conditions. Specifically, the method demonstrated here provides essential information on C. neoformans pathogenesis, such as protein production necessary for virulence and identifies critical host proteins responding to microbial invasion.

Here, we present a protocol to profile the interplay between host and pathogen during infection by mass spectrometry-based proteomics. This protocol uses label-free quantification to measure changes in protein abundance of both host (e.g., macrophages) and pathogen (e.g., Cryptococcus neoformans) in a single experiment.

无标签定量蛋白经济学工作流程，用于发现驱动的宿主-病原体交互

The technological achievements of mass spectrometry (MS)-based quantitative proteomics opens many undiscovered avenues for analyzing an organism&#8217;s ...

Two-Dimensional Gel Electrophoresis

Two-dimensional gel electrophoresis (2DGE) is a technique that can resolve thousands of biomolecules from a mixture. This technique involves two distinct separation methods that have been coupled together: isoelectric focusing (IEF) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). This physically separates compounds across two axes of a gel by their isoelectric points (an electrochemical property) and their molecular weights.
The procedure in this video covers the main concepts of 2DGE and a general procedure for characterizing the composition of a complex protein solution. Three examples of this technique are shown in the applications section, including biomarker detection for disease initiation and progress, monitoring treatment in patients, and the study of proteins following posttranslational modification (PTM).
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Two-dimensional, or 2D, gel electrophoresis is a technique utilizing two distinct separation methods which can separate thousands of proteins from a single mixture. One of the techniques, SDS-PAGE or sodium dodecyl sulfate polyacrylamide gel electrophoresis, cannot fully separate complex mixtures alone. 2D gel electrophoresis couples the SDS-PAGE to a second method, isoelectric focusing or IEF, which separates based on isoelectric points, allowing for the resolution of potentially all proteins in a cell lysate. This video will show the principles of 2D gel electrophoresis, a general procedure, and some of its biomedical applications.
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2D gel electrophoresis begins with IEF as the first dimension. Every protein has a pH value, called the isoelectric point or pI, where the net charge is zero. When a protein is subjected to an electric field, it will move toward the electrode with opposite charge. Samples of interest are loaded onto immobilized pH gradient, or IPG, strips which have embedded ampholytes, molecules containing both acidic and basic groups. An electric field is then applied to the pH gradient strip, causing the proteins to migrate until they reach the pH value matching their pI, where they lose their net charge.
Prior to running the second-dimension, the embedded proteins are treated with SDS, denaturing them and providing a uniform negative charge. Once completed, the IPG strips are placed onto a polyacrylamide gel. An applied electric field draws the proteins toward the anode, with larger proteins moving more slowly through the gel.
Once the protein mixture has been separated according to pI and molecular weight, the proteome map is visualized using stains, and proteins of interest are identified.
Now that we have discussed the principles of 2D gel electrophoresis, let&#39;s go over a typical laboratory procedure.
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Before the experiment can be performed, the proteins must be solubilized into media. Solubilization of the sample is achieved by de-aggregating proteins with a combination of chaotropic agents for disrupting hydrogen-bonding interactions, nonionic detergents to prevent altering of the proteins&#39; charge, reducing agents to break disulfide bonds, and buffers. To remove interfering abundant proteins and other molecules, the material is sequentially extracted by centrifugation, and collection of the resulting pellet; followed by treatment with endonuclease, an enzyme used to consume any DNA that would interfere with the experiment.
Once the proteins have been solubilized, IPG strips are prepared by rinsing with a strip-cleaning solution and left upside-down to dry. Each strip is then assigned a strip holder number. Once ready, the cellular extract is loaded onto the strips in a slow, sliding motion from the negative to the positive end. For the purpose of rehydration, damp blotting paper is placed on top of the electrode and under the gel strips; the IPG strips are then lined onto the IEF instrument. A high electric current is applied, and the proteins begin to migrate.
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Following completion of the first dimension, the gel for SDS-PAGE is prepared in a casting apparatus. The IPG strips are treated by placing them face down in SDS-containing equilibration buffer. The electrophoresis unit is readied with the addition of electrophoresis buffer. The treated IPG strips are collected using tweezers, placed on top of the gel plates, and sealed with agarose-sealing solution. A voltage source then applies an electric field, which is held until the fastest-moving proteins are 1 cm from the bottom of the gel.
After completion of the electrophoresis, the proteins must be visualized. Traditionally this is performed by staining with Coomassie blue or silver nitrate. Proteins of interest may be transferred from the gel, and analyzed by Western blot analysis.
A second identification approach involves excision of the proteins from the gel, digesting them, than analyzing them by mass spectrometry.
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Now that we&#39;ve reviewed a procedure, let&#39;s look at some of the uses for 2D gel electrophoresis.
One of the most common uses for this technique is the identification of molecules involved in disease initiation and progression. 2D gel electrophoresis, coupled with mass spectrometry, can detect the up- or down-regulation of specific proteins in diseased areas in comparison to healthy ones.
Additionally, 2D gel electrophoresis is useful in following the progress of patients&#39; response to a potential therapeutic drug. Specimens may be taken from patients at various timepoints following administration of the treatment. In this way, 2D gel electrophoresis coupled with Western blot or mass spectrometry analysis, can detect proteins associated with negative responses such as inflammation; or the absence of proteins in an alleviated state.
Another use for 2D gel electrophoresis is in the study of protein structure and function following posttranslational modification, or PTM, which are additions to proteins following their translation from mRNA. PTM&#39;s can regulate a variety of functions, including protein signaling, gene expression, or cause oxidative damage. 2D gel electrophoresis is sensitive to modifications such as methylation or acetylation, which can cause a shift in pI as well as the molecular weight.
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You&#39;ve just watched JoVE&#39;s video on 2D gel electrophoresis. This video described the principles of the technique, a typical experimental procedure, and several of its applications in the field of biomedicine.
Thanks for watching!
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二维凝胶电泳

Two-dimensional gel electrophoresis (2DGE) is a technique that can resolve thousands of biomolecules from a mixture. This technique involves two distinct ...