| 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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| Quantification of Methylglyoxal Levels in Cowpea Leaves in Response to Cowpea Aphid Infestation
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Author:
Date:
2020-10-20
[Abstract] Aphids are a serious pest of crops across the world. Aphids feed by inserting their flexible hypodermal needlelike mouthparts, or stylets, into their host plant tissues. They navigate their way to the phloem where they feed on its sap causing little mechanical damage to the plant. Additionally, while feeding, aphids secrete proteinaceous effectors in their saliva to alter plant metabolism and disrupt plant defenses to gain an advantage over the plant. Even with these arsenals to overcome plant responses, plants have evolved ways to detect and counter with defense responses to curtail aphid infestation. One of such response of cowpea to cowpea aphid infestation, is accumulation of the metabolite methylglyoxal. Methylglyoxal is an α,β-dicarbonyl ketoaldehyde that is toxic at high ...
[摘要] [摘要] 蚜虫是世界各地农作物的严重害虫。蚜虫通过将它们柔软的针状口器插入宿主植物组织中取食。它们会进入韧皮部，在那里吃韧皮部的汁液，对植物的机械损伤很小。此外，蚜虫在进食时，会在唾液中分泌蛋白质效应器来改变植物的新陈代谢，破坏植物的防御，从而获得对植物的优势。即使有了这些武器来克服植物的反应，植物也进化出了探测和反击的方式，以减少蚜虫的侵扰。豇豆蚜虫感染的这种反应之一是代谢物甲基乙二醛的积累。甲基乙二醛是一种对、对二羰基酮醛，在高浓度下有毒。在暴露于许多不同的非生物和生物胁迫后，甲基乙二醛水平略有上升，并已被证明在低水平上作为一种新兴的防御信号分子。在此，我们描述了一种在豇豆蚜虫感染后，利用高氯酸提取工艺在豇豆叶片中测量甲基乙二醛的方案。将提取的上清液用碳酸钾中和，丙酮醛与N-乙酰- l-半胱氨酸反应量化，生成N-乙酰- s -(1-羟基-2-oxo-prop-1-yl)半胱氨酸，并进行分光光度定量。
[背景] ...
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