| Novel Protein-oligonucleotide Conjugation Method Involving a High-affinity Capture HaloTag
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Author:
Date:
2020-09-20
[Abstract] Highly sensitive quantitative protein profiling can play a key role in the early diagnosis of diseases, such as autoimmune diseases and cancer. We developed a modified protein-oligonucleotide conjugation method termed HaloTag-mediated barcoding, for quantifying protein molecules at a higher sensitivity than conventional protein quantification methods. This novel and efficient conjugation method can be used to prepare HaloTag-barcoded proteins using a click chemistry-based labeling technique. Here, we describe the preparation of protein-DNA complexes and detection of protein-protein interactions which can be used in a HaloTag protein barcode assay to detect an antibody. The protocol includes procedures for preparing the ligand-oligonucleotide complex, plasmid DNA preparation for protein ...
[摘要] [摘要 ] 高灵敏度的定量蛋白质谱分析可以在疾病的早期诊断中发挥关键作用，例如自身免疫性疾病和癌症。我们开发了一种改良的蛋白质-寡核苷酸缀合方法，称为HaloTag 介导的条形码，用于以比常规蛋白质定量方法更高的灵敏度来定量蛋白质分子。可以使用这种基于点击化学的标记技术，将这种新颖而有效的结合方法用于制备HaloTag 条形码蛋白。在这里，我们描述了可在HaloTag中使用的蛋白质-DNA复合物的制备和蛋白质-蛋白质相互作用的检测 蛋白质条形码检测以检测抗体。该方案包括制备配体-寡核苷酸复合物的程序，用于蛋白质表达的质粒DNA制备以及蛋白质-寡核苷酸复合物的制备。所描述的基于点击反应的方案简化了常规胺-酯反应方法，该方法需要色谱纯化的额外步骤。
[背景 ] 蛋白质分子可通过常规实验进行定量酶联免疫吸附测定法，western印迹和质谱的方法，例如。这些常规的定量蛋白质谱分析技术涉及使用校准曲线进行相对测量，而没有考虑DNA扩增的高灵敏度，这限制了蛋白质本身绝对量的检测。化学蛋白质组学成为可能多重测定我n中的相对定量方式，例如串联质量标签标记方法加上质谱（汤普森等人，2003 ）。蛋白质条形码技术与下一代测序技术相结合已经可以识别目标蛋白质分子。这些方法包括CITE- SEQ ，的Ab- SEQ 和L1 BRA- SEQ ...
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| Identification of RNA-binding Proteins
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Author:
Date:
2016-09-05
[Abstract] This protocol describes the extraction of RNA-binding proteins (RBPs) from cell lysates. In order to pull down target RBPs, 5-bromo-UTP (BrUTP)-incorporated RNA probes are used, which are generated by in vitro transcription. The schematic diagram (Flowchart) with procedure is indicated (Figure1 and Figure 2).
Figure 1. Schematic diagram of procedure (A-H). Flow chart of experimental procedure is indicated at A-H.
图1.程序示意图（AH）。实验程序的流程图在AH指示。
图2.质粒的线性化 限制性酶。在与其克隆元件相邻的限制性位点切割质粒。
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| Root-knot Nematode Penetration and Sclareol Nematicidal Activity Assays
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Author:
Date:
2016-06-20
[Abstract] Plant parasitic nematodes parasitize roots and/or stems of various plants thereby inhibiting absorption of nutrients and moisture. In particular, root-knot nematodes (RKN) are a group of the most devastating pests. Various techniques, such as soil sterilization, cultivation of resistant crops, and chemical application, have been developed to control damage caused by RKN. Among these techniques, diminish by chemicals that induce or activate host defense to RKN is an attractive method because of its potential to reduce the environmental burden caused by crop protection. Sclareol, a diterpene, was identified as a chemical that induces resistance to RKN (Fujimoto et al., 2015). Here we provide a protocol for assessing the impact of sclareol on the penetration of RKNs into tomato and Arabidopsis ...
[摘要] 植物寄生线虫寄生于各种植物的根和/或茎，从而抑制营养物和水分的吸收。 特别地，根结线虫（RKN）是最具破坏性的害虫的组。 已经开发了各种技术，例如土壤灭菌，抗性作物的栽培和化学施用，以控制由RKN引起的损害。 在这些技术中，诱导或激活RKN的宿主防御的化学物质的减少是有吸引力的方法，因为其具有减少作物保护所引起的环境负担的潜力。 Sclareol，一种二萜，被鉴定为诱导对RKN的抗性的化学物质（Fujimoto等人，2015）。 在这里我们提供了评估香紫苏醇对RKNs对番茄和拟南芥根的渗透以及化学物对线虫的直接杀线虫影响的影响的方案。 该方案可用于其它线虫抗性诱导化学品。
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