Moser Annette C, White Benjamin, Kovacs Frank A
Chemistry Department, University of Nebraska at Kearney, 905 West 25th Street, Kearney, NE, 68849-1150, USA,
Methods Mol Biol. 2014;1129:423-34. doi: 10.1007/978-1-62703-977-2_30.
Affinity chromatography is one way to measure the binding constants of a protein-ligand interaction. Here we describe a method of measuring a binding constant using Ni-NTA resin to immobilize a His-tagged enzyme and the method of frontal analysis. While other methods of immobilization are possible, using the strong affinity interaction between His-tagged proteins and Ni-NTA supports results in a fast, easy, and gentle method of immobilization. Once the affinity support is created, frontal analysis can be used to measure the binding constant between the protein and various analytes.
亲和层析是测量蛋白质-配体相互作用结合常数的一种方法。在此，我们描述一种使用镍-次氮基三乙酸（Ni-NTA）树脂固定化带有组氨酸标签的酶来测量结合常数的方法以及前沿分析法。虽然也可以采用其他固定化方法，但利用带有组氨酸标签的蛋白质与Ni-NTA载体之间的强亲和相互作用可得到一种快速、简便且温和的固定化方法。一旦创建了亲和载体，就可以使用前沿分析法来测量蛋白质与各种分析物之间的结合常数。
