Büssow K, Cahill D, Nietfeld W, Bancroft D, Scherzinger E, Lehrach H, Walter G
Max Planck Institute for Molecular Genetics, Ihnestr. 73, D-14195 Berlin, Germany.
Nucleic Acids Res. 1998 Nov 1;26(21):5007-8. doi: 10.1093/nar/26.21.5007.
We have developed a technique to establish catalogues of protein products of arrayed cDNA clones identified by DNA hybridisation or sequencing. A human fetal brain cDNA library was directionally cloned in a bacterial vector that allows IPTG-inducible expression of His6-tagged fusion proteins. Using robot technology, the library was arrayed in microtitre plates and gridded onto high-density in situ filters. A monoclonal antibody recognising the N-terminal RGSH6sequence of expressed proteins (RGS.His antibody, Qiagen) detected 20% of the library as putative expression clones. Two example genes, GAPDH and HSP90alpha, were identified on high-density filters using DNA probes and antibodies against their proteins.
我们已经开发出一种技术，用于建立通过DNA杂交或测序鉴定的阵列cDNA克隆的蛋白质产物目录。一个人类胎儿脑cDNA文库被定向克隆到一个细菌载体中，该载体允许IPTG诱导表达His6标签融合蛋白。利用机器人技术，该文库被排列在微量滴定板中，并网格化到高密度原位滤膜上。一种识别表达蛋白N端RGSH6序列的单克隆抗体（RGS.His抗体，Qiagen）检测到20%的文库为推定表达克隆。使用针对两种示例基因GAPDH和HSP90alpha的DNA探针及其蛋白质的抗体，在高密度滤膜上鉴定出了这两个基因。
