Orthwein Tim, Huergo Luciano F, Forchhammer Karl, Selim Khaled A
Organismic Interactions Department, Interfaculty Institute for Microbiology and Infection Medicine, Cluster of Excellence 'Controlling Microbes to Fight Infections', Tübingen University, Auf der Morgenstelle 28, 72076 Tübingen, Germany.
Setor Litoral, Federal University of Paraná (UFPR), Matinhos, Brazil.
Bio Protoc. 2021 Sep 5;11(17):e4152. doi: 10.21769/BioProtoc.4152.
Biolayer interferometry (BLI) is an emerging analytical tool that allows the study of protein complexes in real time to determine protein complex kinetic parameters. This article describes a protocol to determine the K of a protein complex using a 6×His tagged fusion protein as bait immobilized on the NTA sensor chip of the FortéBio Octet K2 System (Sartorius). We also describe how to determine the half maximal effective concentration (EC, also known as IC for inhibiting effectors) of a metabolite. The complete protocol allows the determination of protein complex K and small molecular effector EC within 8 h, measured in triplicates. Graphic abstract: (Middle, top) Exemplary result of the BLI measurement using Octet (Raw Data). Wavelength shift (Δλ) against time. (A) Baseline 1. Baseline measurement. When the sensor is equilibrated in the kinetics buffer. The light is reflected with no difference. (B) Load. The his-tagged proteins (ligand) are loaded onto the sensor surface. The light is reflected with a shift of the wavelength. (C) Baseline 2. The loaded sensor is equilibrated in the kinetics buffer. No further wavelength shift appears. (D) Association. The loaded sensor is dipped into the analyte solution. The analyte binds to the immobilized ligand along with an increased wavelength shift. (E) Dissociation. Afterward, the sensor is dipped again into the kinetics buffer without the analyte. Some analyte molecules dissociate. The wavelength shift decreases. (Subfigures A-E) The left side shows the position of the sensor during the measurement seen in the representative BLI measurement, marked with the figure label. The right side shows the light path in the sensor. Black waves represent the light emitted to the sensor surface. The red waves show the light reflected from the sensor surface back to the detector.
生物层干涉术（BLI）是一种新兴的分析工具，可实时研究蛋白质复合物，以确定蛋白质复合物的动力学参数。本文介绍了一种使用固定在福泰生物Octet K2系统（赛多利斯）的NTA传感器芯片上的6×组氨酸标签融合蛋白作为诱饵来测定蛋白质复合物Kd的方法。我们还描述了如何测定代谢物的半数最大效应浓度（EC50，对于抑制性效应物也称为IC50）。完整的方法可在8小时内完成蛋白质复合物Kd和小分子效应物EC50的测定，测量一式三份。图形摘要：（中间，顶部）使用Octet进行BLI测量的示例性结果（原始数据）。波长偏移（Δλ）随时间变化。（A）基线1。基线测量。当传感器在动力学缓冲液中平衡时。光反射无差异。（B）加载。组氨酸标签蛋白（配体）加载到传感器表面。光反射时波长发生偏移。（C）基线2。加载后的传感器在动力学缓冲液中平衡。不再出现波长偏移。（D）结合。加载后的传感器浸入分析物溶液中。分析物与固定化配体结合，同时波长偏移增加。（E）解离。之后，传感器再次浸入不含分析物的动力学缓冲液中。一些分析物分子解离。波长偏移减小。（子图A - E）左侧显示在代表性BLI测量中测量期间传感器的位置，标有图标签。右侧显示传感器中的光路。黑色波浪表示发射到传感器表面的光。红色波浪显示从传感器表面反射回检测器的光。
