| Identifying Protein Interactions with Histone Peptides Using Bio-layer Interferometry
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Author:
Date:
2018-09-20
[Abstract] Histone post-translational modifications (PTMs) regulate numerous cellular processes, including gene transcription, cell division, and DNA damage repair. Most histone PTMs affect the recruitment or exclusion of reader proteins from chromatin. Here, we present a protocol to measure affinity and interaction kinetics between histone peptides and the recombinant protein using Bio-layer interferometry.
[摘要] 组蛋白翻译后修饰（PTM）调节许多细胞过程，包括基因转录，细胞分裂和DNA损伤修复。 大多数组蛋白PTM影响从染色质中募集或排除读取蛋白。 在这里，我们提出了一个协议，使用生物层干涉测量法测量组蛋白肽和重组蛋白之间的亲和力和相互作用动力学。
【背景】真核染色质结构大致分为常染色质和异染色质（Cheung和Lau，2005），异染色质结构根据组蛋白翻译后修饰（PTM）的组合进一步细分。这些PTM不仅改变染色质构象，还在基因表达和蛋白质募集中建立直接调节作用（Felsenfeld和Groudine，2003; Allshire和Madhani，2017）。组蛋白PTM的无数组合 - 包括乙酰化，磷酸化，甲基化，泛素化，生物素化，SUMO化和脯氨酸异构化，统称为“组蛋白标记” - 可以被发现，特别是在从核小体核心突出的非结构化N末端尾部（ Guetg和Santoro，2012）。这些PTM通过不同“读者”或效应蛋白的活动调节许多细胞过程，包括基因转录，细胞分裂和DNA损伤修复（Suganuma和Workman，2011）（Musselman et al。， 2012）。因此，已经做出很大努力来识别读者的组蛋白修饰。
使用常规方法（例如，表面等离子体共振[SPR]和SPR成像[SPRi]生物传感器）研究读取蛋白与其靶蛋白PTM之间的相互作用通常需要大量底物或复杂的多步实验方法并且由于各种方法特定的限制而变得复杂。这些问题排除了量化相互作用强度的简便性和准确性（Phizicky和Fields，1995; ...
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| Kinetic Analysis of Monoclonal Antibody Binding to HIV-1 gp120-derived Hyperglycosylated Cores
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Author:
Date:
2015-10-05
[Abstract] Kinetic analysis of antibodies is one of the important studies for characterization of antibodies and screening of ligands. In our recent study (Ingale et al., 2014), we compared the antigenic profiles and binding characteristics of four HIV-1 envelope glycoprotein (Env) core immunogens using multiple monoclonal antibodies by Bio-Layer Light Interferometry (BLI). This technology enables real-time analysis of interactions on the surface of a fiber optic biosensor by accurately measuring kinetic constants such as Ka, Kd, and KD in a 96-well format.
[摘要] Kinetic analysis of antibodies is one of the important study for characterization of antibodies and screening of ligands. In our recent study1, we compared the antigenic profiles and binding characteristics of four HIV-1 envelope glycoprotein (Env) core immunogens using multiple monoclonal antibodies by Bio-Layer Light Interferometry (BLI). This technology enables real-time analysis of interactions on the surface of a fiber optic biosensor by accurately measuring kinetic constants such as Ka, Kd, and KD in a 96-well format.
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| Determination of Protein-DNA (ZMYND11-DNA) Interaction by a Label-Free Biolayer Interferometry Assay
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Author:
Date:
2015-02-20
[Abstract] This protocol describes a robust technique for the measurement of ZMYND11-DNA interaction by a label-free Biolayer Interferometry (BLI). ZMYND11 is a novel histone reader protein that specifically recognizes H3.3K36me3 via its tandem Bromodomain, zinc-finger and PWWP domain (BP). ZMYND11 links the histone-variant-mediated transcription elongation control to tumour suppression and may therefore represent a novel class of drug targets. Like other PWWP domains, ZMYND11 PWWP domain shows highly positively charged surface and interacts with DNA. Previously reported methods include NMR, FP or ...
[摘要] 该协议描述了用于通过无标记生物分子干涉测量法（BLI）测量ZMYND11-DNA相互作用的鲁棒技术。 ZMYND11是一种新型组蛋白阅读器蛋白，通过其串联溴结构域，锌指和PWWP结构域（BP）特异性识别H3.3K36me3。 ZMYND11将组蛋白变体介导的转录伸长控制与肿瘤抑制联系起来，因此可能代表一种新型的药物靶标。像其他PWWP域一样，ZMYND11 PWWP域显示高度带正电荷的表面，并与DNA相互作用。先前报道的方法包括NMR，FP或EMSA。生物层干涉法（BLI）是用于分析各种生物分子相互作用（例如蛋白质 - 蛋白质和蛋白质-DNA结合）的新兴技术。 BLI允许实时监测生物分子之间的相互作用，而不需要具有酶，荧光或放射性标记的试剂。该技术基于当材料结合到光纤的尖端时从光纤的表面反射的光的干涉图案的变化。该技术代表了诸如表面等离子体共振等技术的替代方法，提供了一种简单的平台，其能够在不使用流动池的情况下实现生物分子相互作用的无标记监测。无标记的生物传感器方法提供关于结合，动力学，浓度和相互作用的亲和力的信息。
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