| Combining Gel Retardation and Footprinting to Determine Protein-DNA Interactions of Specific and/or Less Stable Complexes
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Author:
Date:
2020-12-05
[Abstract] DNA footprinting is a classic technique to investigate protein-DNA interactions. However, traditional footprinting protocols can be unsuccessful or difficult to interpret if the binding of the protein to the DNA is weak, the protein has a fast off-rate, or if several different protein-DNA complexes are formed. Our protocol differs from traditional footprinting protocols, because it provides a method to isolate the protein-DNA complex from a native gel after treatment with the footprinting agent, thus removing the bound DNA from the free DNA or other protein-DNA complexes. The DNA is then extracted from the isolated complex before electrophoresis on a sequencing gel to determine the footprinting pattern. This analysis provides a possible solution for those who have been unable to use ...
[摘要] [摘要] DNA足迹是研究蛋白质-DNA相互作用的经典技术。但是，如果蛋白质与DNA的结合较弱，蛋白质的脱落速率较快，或者形成了几种不同的蛋白质-DNA复合物，则传统的足迹方案可能会失败或难以解释。我们的协议不同于传统的足迹协议，因为它提供了一种在使用足迹剂处理后从天然凝胶中分离蛋白质-DNA复合物的方法，从而从游离DNA或其他蛋白质-DNA复合物中去除了结合的DNA。然后从分离的复合物中提取DNA，然后在测序凝胶上电泳以确定印迹模式。该分析为无法使用传统足迹法确定蛋白质与DNA接触的人提供了可能的解决方案。
[背景]核酸酶/化学足迹是一个典型的方法来探测蛋白质-DNA相互作用（腊士和施米茨，1978;萨瑟-德怀特和Gralla，1989;汉普等人，2007） ...
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| Single-cell Visualization of Chromosome Transcriptional Territories by RNA-paint
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Author:
Date:
2016-09-05
[Abstract] We developed a FISH-based method to directly assess chromosome-wide transcriptional activity, thereby enabling the visualization of the actively transcribed fraction of a chromosome at the single-cell level. We applied this method to probe the activity of X-chromosomes and its instability in the context of human embryonic stem cells and cancer cells.
[摘要] 我们开发了基于FISH的方法，以直接评估染色体宽转录活性，从而使单细胞水平的染色体的积极转录部分的可视化。 我们应用这种方法探测X染色体的活动及其在人类胚胎干细胞和癌细胞的上下文中的不稳定性。
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| Combined in situ Hybridization/Immunohistochemistry (ISH/IH) on Free-floating Vibratome Tissue Sections
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Author:
Date:
2014-09-20
[Abstract] In situ hybridization and immunostaining are common techniques for localizing gene expression, the mRNA and protein respectively, within tissues. Both techniques can be applied to tissue sections to achieve similar goals, but in some cases, it is necessary to use them together. For example, complement C1q is a secreted protein complex that can target the innate immune response during inflammation. Complement has been found to be elevated early and before severe neurodegeneration in several disease models. Thus, complement may serve as an important marker for disease progression and may contribute to the pathology under certain conditions. Since complement is a secreted complex, immunostaining for C1q does not necessarily reveal where compliment is produced. In situ ...
[摘要] 原位杂交和免疫染色是分别定位组织内基因表达，mRNA和蛋白质的常用技术。两种技术可以应用于组织切片以实现类似的目标，但是在一些情况下，有必要一起使用它们。例如，补体C1q是可以靶向炎症期间的先天免疫应答的分泌的蛋白复合物。已经发现补体在几种疾病模型中早期升高和严重神经变性之前升高。因此，补体可以作为疾病进展的重要标志物，并且可能有助于在某些条件下的病理学。由于补体是分泌的复合物，C1q的免疫染色不一定揭示产生补偿的位置。补体成分，C1q a，b或c mRNA的原位杂交是理想的标记组织中产生补体的细胞。原位杂交可以与细胞类型特异性免疫染色偶联以准确鉴定所涉及的细胞类型。蛋白质定位和mRNA定位一起可以揭示关于疾病组织内补体产生和补体靶细胞之间的关系的细节。在这里我们概述在同一组织切片上的组合原位杂交和免疫染色的步骤。这里概述的协议已设计用于检测小鼠脑神经元和小胶质细胞中的补体C1q。
这里提供了两种组合ISH/IH的方法。在第一个实施例中，C1q mRNA的原位杂交与使用Calbindin-D28K抗体的Purkinje神经元细胞体的荧光检测一起进行。在第二个实施例中，使用CD68抗体与小胶质细胞的3,3'-二氨基联苯胺（DAB）一起进行C1q ...
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