Name: the use of reverse phase protein arrays (rppa) to explore protein expression variation within individual renal cell cancers
Uploaded: 2013-01-22T13:00:00
Description: Watch this Scientific Journal Video about The Use of Reverse Phase Protein Arrays RPPA to Explore Protein Expression Variation within Individual Renal Cell Cancers at JoVE.com

作者FCO，DF，JN，DJH和GDS的工作上面提到的由由英国癌症研究中心实验癌症医学中心的首席科学家办公室，项目编号：ETM37和支持。 AL的工作是由皇家爱丁堡外科的护理和治疗癌症和医学研究理事会罗伯逊信托，梅尔维尔信托。 IO的支持，玛丽·居里行动和英国医学研究理事会共同资助的英国皇家学会的爱丁堡苏格兰政府奖学金。作者想感谢SCOTRRCC共同申请人及其合作者提出了有用的讨论本文讨论的主题。

1。形态学和分子肿瘤的异质性鉴定<ol><li>肿瘤从-80℃的冰箱中取出并保持在干冰上。 </li><li>划分成部分的约1厘米3的肿瘤。映射每个肿瘤的部分彼此相对，并与一个唯一的名称标签的原始位置。将样品存放在个别的离心管中，在-80℃，直到准备使用。 </li><li>涂层样品在OCT和减少在低温恒温器在-22°C。 </li><li>样品染色，苏木精和曙红counterstaining方法。 </li><li>显微镜分析冰冻切片，以确?...

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RPPA这里介绍的方法代表一个高吞吐量的替代广泛使用，但相对较低的吞吐量的蛋白质分析的免疫印迹技术。该方法允许数百个样品是半定量的分析和比较，同时允许在很宽的选择的细胞系和组织样品中的关键蛋白的直接比较。复用与不同的抗体物种进一步增加电源的技术允许同时使用多种抗体。这里介绍的例子强调的能力RPPA在研究肿瘤的异质性和异质性靶向治疗的效果。 ?...

<table border="1"><tbody><tr><td> 的试剂的名称 </td><td> 公司 </td><td> 目录编号 </td></tr><tr><td>抑肽酶</td><td>西格玛</td><td> A6279 </td></tr><tr><td>磷酸酶抑制剂的鸡尾酒2 </td><td>西格玛</td><td> P5726 </td></tr><tr><td>磷酸酶抑制剂的鸡尾酒3 </td><td>西格玛</td><td> P0044 </td></tr><tr><td>蛋白酶抑制剂混合物</td><td>罗氏公司</td><td> 11836153001 </td></tr><tr><td>的Triton X-100的</td><td>的Triton-X </td><td> T8787 </td></tr><tr><td> LI-COR Odyssey封闭缓冲液</td><td> LI-COR </td><td> 927-40000 </td></tr><tr><td> TissueLyser </td><td> Qiagen公司</td><td> 85600 </td></tr><tr><td>微网II机器人去污剂</td><td> Biorobotics </td><td></td></tr><tr><td> FastFrame“四个海湾幻灯片夹</td><td> Whatman公司</td><td> 10486001 </td></tr><tr><td> FAST幻灯片 - 2 - 垫</td><td> Whatman公司</td><td> 10485317 </td></tr><tr><td> IRDye 680LT山羊抗鼠IgG </td><td> LICOR </td><td> 926-68020 </td></tr><tr><td> IRDye 800CW的山羊抗兔IgG </td><td> LICOR </td><td> 926-32211 </td></tr></tbody></table>

the use of reverse phase protein arrays (rppa) to explore protein expression variation within individual renal cell cancers

目前还没有治愈的疾病治疗的转移性肾透明细胞癌，最常见的变种。这种治疗性的一个关键因素被认为是分子复杂性的疾病1。如酪氨酸激酶抑制剂（TKI），舒尼替尼靶向治疗已被使用，但只有40％的病人会响应，这些患者绝大多数复发1年以内2。因此在肾细胞癌患者的内在和获得性耐药的问题是高度相关的。 为了研究抗酪氨酸激酶抑制剂，制定有效的个性化治疗的最终目标，有针对性的治疗后，在规定的顺序组织是必需的，已被证明是成功的做法，在慢性粒细胞白血病4。然而，这样的策略在肾细胞癌的应用是复杂的，高层次的b超视距间和肿瘤的异质性，这是一个功能的肾细胞癌5,6以及其他实体瘤7。即使在建立类似的介绍，肿瘤的分期，分级的患者Intertumoral异质性由于转录和遗传差异的。此外，它是明确的，也有很大的形态（肿瘤内）的异质性RCC，这很可能代表更大的分子异质性。 RCC肿瘤的综合形态分析和Fuhrman分级详细的映射和分类，用户可以通过选择有代表性的地区蛋白质组学分析。 基于蛋白质的分析的RCC 8是有吸引力的，但由于其广泛的可用性，在病理实验室，它的应用可以是有问题的，因为供应量有限，特异性抗体9。由于斑点印迹性质的反相蛋白质阵列（RPPA），抗体的特异性必须Ë预先验证的，使用的抗体，这种严格的质量控制是非常重要的。尽管这一局限斑点印迹格式不允许检测的小型化，使数百个样品用于印刷到一个单一的硝酸纤维素滑动。然后，印刷幻灯片可以用类似的方式进行分析，Western印迹分析与特定目标初级抗体和荧光标记的第二抗体的使用，允许复用。在所有的样品在幻灯片上的差异蛋白质表达，然后可以同时分析在一个更具成本效益和高通量的方式进行比较的相对水平的荧光。

一个的扫描RPPA滑动一个例子可以看出，在图4（i）与680和800 nm的通道所示。由图4（ⅱ）中的波长，使分离的图像每个焊盘上的RPPA滑动以进行分析，并确定图4（ⅲ）中的单独的蛋白表达。正如图4可以看出，在（iii）该个别蛋白质表达的整个样品与凝溶胶蛋白具有高的表达水平横跨幻灯片cMYC具有低得多的蛋白表达相比是独特的，但仍?...

Watch this Scientific Journal Video about The Use of Reverse Phase Protein Arrays RPPA to Explore Protein Expression Variation within Individual Renal Cell Cancers at JoVE.com

The Use of Reverse Phase Protein Arrays RPPA to Explore Protein Expression Variation within Individual Renal Cell Cancers

RPPA支持数百个样品，印在硝酸纤维素幻灯片同时进行审讯，用荧光标记的抗体蛋白的表达。这项技术已被应用在肾透明细胞癌的药物治疗的非均质性研究的效果。

使用反时相蛋白的阵列（RPPA）探索在个人的肾细胞癌蛋白表达的变化

Currently there is no curative treatment for metastatic clear cell renal cell cancer, the commonest variant of the disease. A key factor in this treatment ...

Research

JoVE Journal

Medicine

The Use of Reverse Phase Protein Arrays (RPPA) to Explore Protein Expression Variation within Individual Renal Cell Cancers

Use of a Hanging-weight System for Isolated Renal Artery Occlusion

一个悬重系统使用隔离肾动脉闭塞

In hospitalized patients, over 50% of cases of acute kidney injury (AKI) are caused by renal ischemia 1-3. A recent study of hospitalized patients ...

科研

医学

Heterogeneity Mapping of Protein Expression in Tumors using Quantitative Immunofluorescence

在使用定量免疫肿瘤的异质性蛋白表达的映射

Morphologic heterogeneity within an individual tumor is well-recognized by histopathologists in surgical practice. While this often takes the form of ...

Production and Detection of Reactive Oxygen Species (ROS) in Cancers

Production and Detection of Reactive Oxygen Species ROS in Cancers

在癌症的生产和检测活性氧（ROS）的

Reactive oxygen species include a number of molecules that damage DNA and RNA and oxidize proteins and lipids (lipid peroxydation). These reactive ...

Biomarkers in an Animal Model for Revealing Neural, Hematologic, and Behavioral Correlates of PTSD

在动物模型揭示神经，血液，创伤后应激障碍和行为相关因素的生物标志物

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for ...

Purification and Aggregation of the Amyloid Precursor Protein Intracellular Domain

淀粉样前体蛋白胞内结构域的分离纯化和聚集

Amyloid precursor protein (APP) is a type I transmembrane protein associated with the pathogenesis of Alzheimer's disease (AD). APP is characterized by a ...

Microarray-based Identification of Individual HERV Loci Expression: Application to Biomarker Discovery in Prostate Cancer

应用生物标志物发现在前列腺癌：个别赫尔维位点表达微阵列鉴定

The prostate-specific antigen (PSA) is the main diagnostic biomarker for prostate cancer in clinical use, but it lacks specificity and sensitivity, ...

Three-Dimensional Culture Assay to Explore Cancer Cell Invasiveness and Satellite Tumor Formation

三维培养试验探索肿瘤细胞浸润和卫星肿瘤形成

Mammalian cell culture in monolayers is widely used to study various physiological and molecular processes. However, this approach to study growing cells ...

An Efficient Sieving Method to Isolate Intact Glomeruli from Adult Rat Kidney

一种从成年大鼠肾脏中分离完整型肾小球的有效筛分方法

Preservation of glomerular structure and function is pivotal in the prevention of glomerulonephritis, a category of kidney disease characterized by ...

Precision Implementation of Minimal Erythema Dose (MED) Testing to Assess Individual Variation in Human Inflammatory Response

Precision Implementation of Minimal Erythema Dose MED Testing to Assess Individual Variation in Human Inflammatory Response

精确实施最小红斑剂量（MED）测试，以评估人体炎症反应中的个体变异

Minimal erythema dose (MED) testing is frequently used in clinical settings for determining the smallest amount of ultraviolet (UV) irradiation necessary ...

Direct Drug Delivery to Kidney via the Renal Artery

通过肾脏动脉直接向肾脏输送药物

There is a need for directed injections to enable increased and specific renal exposure for efficient evaluation of drug targets in the renal research ...