Miskimins W K, Roberts M P, McClelland A, Ruddle F H
Proc Natl Acad Sci U S A. 1985 Oct;82(20):6741-4. doi: 10.1073/pnas.82.20.6741.
We describe a procedure for detecting high-affinity, sequence-specific DNA-binding proteins from crude nuclear extracts. The technique utilizes electrophoretic transfer of NaDodSO4/PAGE-fractionated proteins onto nitrocellulose filters. Incubation of the filters with a 5% (wt/vol) solution of nonfat dry milk effectively blocks nonspecific and low-affinity DNA-binding sites. Incubation of the blocked filters with radiolabeled DNA under optimal binding conditions and subsequent autoradiography reveals high-affinity DNA-protein interactions. We have used this procedure to identify proteins that bind specifically to the promoter region of the transferrin receptor gene.
我们描述了一种从粗核提取物中检测高亲和力、序列特异性DNA结合蛋白的方法。该技术利用将经十二烷基硫酸钠/聚丙烯酰胺凝胶电泳（NaDodSO4/PAGE）分离的蛋白质电泳转移到硝酸纤维素滤膜上。用5%（重量/体积）脱脂奶粉溶液孵育滤膜可有效封闭非特异性和低亲和力DNA结合位点。在最佳结合条件下，用放射性标记的DNA孵育封闭后的滤膜，随后进行放射自显影，可揭示高亲和力DNA-蛋白质相互作用。我们已使用该方法鉴定了与转铁蛋白受体基因启动子区域特异性结合的蛋白质。
