Weiner Hendrik, Faupel Thomas, Büssow Konrad
Department of Vertebrate Genomics, Max Planck Institute of Molecular Genetics, Berlin, Germany.
Methods Mol Biol. 2004;264:1-13. doi: 10.1385/1-59259-759-9:001.
This chapter describes the production of a cDNA expression library from human fetal brain, the construction of a high-density protein array from such a library, and two applications to screen the array for binding proteins. After producing the library and decollating the expression clones, one can pick thousands of expression clones with a laboratory robot and can deposit them into microtiter plates in an ordered manner. Such ordered clone libraries are the starting material for the construction of a high-density protein array. This array is constructed by spotting the expression clones onto a protein-binding membrane. Following cell growth and induction of protein expression on the membrane, the cell spots are lysed and their recombinant protein immobilized on the membrane. The so-constructed array carries thousands of proteins without the need to clone, express, and spot individual proteins. Such arrays allow one to screen for numerous protein functions in a high-throughput manner.
本章描述了从人胎脑中制备cDNA表达文库、从该文库构建高密度蛋白质阵列以及两种用于筛选该阵列以寻找结合蛋白的应用。制备文库并分离表达克隆后，可以使用实验室机器人挑选数千个表达克隆，并将它们有序地接种到微孔板中。这种有序的克隆文库是构建高密度蛋白质阵列的起始材料。该阵列通过将表达克隆点样到蛋白质结合膜上构建而成。在膜上细胞生长并诱导蛋白质表达后，细胞点被裂解，其重组蛋白固定在膜上。如此构建的阵列携带数千种蛋白质，无需对单个蛋白质进行克隆、表达和点样。这种阵列使人们能够以高通量方式筛选多种蛋白质功能。
