Jutras Brandon L, Verma Ashutosh, Stevenson Brian
Department of Microbiology, Immunology, and Molecular Genetics, University of Kentucky College of Medicine, Lexington, Kentucky.
Curr Protoc Microbiol. 2012 Feb;Chapter 1:Unit1F.1. doi: 10.1002/9780471729259.mc01f01s24.
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.
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。