利用DNA亲和色谱法/下拉技术鉴定新型DNA结合蛋白。
Identification of novel DNA-binding proteins using DNA-affinity chromatography/pull down.
作者信息
Jutras Brandon L, Verma Ashutosh, Stevenson Brian
机构信息
Department of Microbiology, Immunology, and Molecular Genetics, University of Kentucky College of Medicine, Lexington, Kentucky.
出版信息
Curr Protoc Microbiol. 2012 Feb;Chapter 1:Unit1F.1. doi: 10.1002/9780471729259.mc01f01s24.
Abstract
This units presents methods through which one may isolate and identify novel bacterial DNA-binding proteins. Briefly, the DNA sequence of interest is affixed to beads, and then incubated with bacterial cytoplasmic extract. Washes with buffers containing nonspecific DNA and low-salt concentrations will remove non-adhering and low-specificity DNA-binding proteins, while subsequent washes with higher salt concentrations will elute more specific DNA-binding proteins. Eluted proteins may then be identified by standard proteomic techniques.
摘要
本单元介绍了分离和鉴定新型细菌DNA结合蛋白的方法。简要地说，将感兴趣的DNA序列固定在珠子上，然后与细菌细胞质提取物一起孵育。用含有非特异性DNA和低盐浓度的缓冲液洗涤将去除非粘附性和低特异性的DNA结合蛋白，而随后用较高盐浓度洗涤将洗脱更具特异性的DNA结合蛋白。然后可以通过标准蛋白质组学技术鉴定洗脱的蛋白质。
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