Cold Spring Harb Protoc. 2020 Jun 1;2020(6):102194. doi: 10.1101/pdb.prot102194.
Immobilized metal affinity chromatography (IMAC) is based on the affinity of polyhistidine tracts for divalent metal cations (usually Ni) immobilized as transition metal chelate complexes on a chromatography resin. The main protocol here is optimized for use of Ni-NTA resin to purify soluble 6xHis-tagged proteins by a straightforward batch method during the binding step, followed by gravity flow for washes and elution. This protocol does not require any specialized equipment other than a simple glass or plastic column. IMAC resins can be used in multiple formats, including batch, gravity flow, centrifuge columns, and fast performance liquid chromatography (FPLC) systems. FPLC systems are designed specifically for the chromatographic separations of proteins and other biomolecules. These systems typically contain multiple pumps, an in-line UV absorption monitor, conductivity meter, pH meter, fraction collector, and other options that allow for the simultaneous purification, analysis, and fractionation of the sample. When linked with the appropriate instruments, an FPLC can become a high-precision, automated instrument that separates proteins at a high resolution. An alternative protocol is included here that describes 6xHis-tagged protein purification using FPLC. Procedures for the cleaning and regeneration of the IMAC resin for reuse are also described, and, finally, considerations for storing purified proteins are discussed.
固定化金属亲和层析（IMAC）基于多组氨酸序列与固定在层析树脂上的过渡金属螯合物络合物的二价金属阳离子（通常为 Ni）的亲和力。这里的主要方案经过优化，可用于 Ni-NTA 树脂通过简单的批量方法在结合步骤中纯化可溶性 6xHis 标记的蛋白质，然后通过重力流进行洗涤和洗脱。该方案除了简单的玻璃或塑料柱外，不需要任何特殊设备。IMAC 树脂可用于多种形式，包括批量、重力流、离心柱和快速性能液相色谱（FPLC）系统。FPLC 系统专为蛋白质和其他生物分子的色谱分离而设计。这些系统通常包含多个泵、在线紫外吸收监测器、电导率计、pH 计、馏分收集器和其他选项，可实现样品的同时纯化、分析和馏分。当与适当的仪器连接时，FPLC 可以成为一种高精度、自动化的仪器，可实现蛋白质的高分辨率分离。这里还包含一个替代方案，描述了使用 FPLC 纯化 6xHis 标记的蛋白质。还介绍了用于重复使用的 IMAC 树脂的清洁和再生的程序，最后讨论了储存纯化蛋白质的注意事项。
