非放射性EMSA法检测AP-1蛋白DNA结合活性实验优化

目的探索一种方案简便,仪器要求较低,且有效的非放射性凝胶电泳迁移率（EMSA）的检测方法。方法以AP-1蛋白为实验对象,自制退火缓冲液合成生物素标记AP-1探针,抽提炎症大鼠脊髓背角处核蛋白（无需纯化）,取10μg核蛋白与探针室温反应30 min。小型聚丙烯酰胺非变性凝胶电泳后将探针湿转至正电子加强尼龙膜上,紫外交联,ECL显影。通过正常探针与变性探针的竞争实验,证明方法的可靠性。结果自制退火缓冲液成功合成生物素标记AP-1探针;炎症大鼠脊髓背角处AP-1蛋白与正常探针的结合后产生明显滞后条带,且结合力远高于变异探针。结论优化后的非放射性EMSA方案,有效降低了实验花费和仪器要求,且结果可信。

Optimized non-radioactive EMSA for detecting AP-1 protein DNA binding activity

Objective To explore a lower instrument required, easier program and effective non-radioactive electrophoretic mobility shift assay （EMSA） detection methods. Methods With AP-1 proteins as experimental subject, nuclear protein was extracted from spinal cord dorsal horn of inflammatory rats. 10 μg nuclear protein responded with AP-1 probe 30 min at room temperature, which was synthesized by homemade annealing buffer synthetic. After non-denaturing polyacrylamide gel elec- trophoresis,probe was transferred to the positron reinforced nylon membrane, UV cross-linking and exposured by ECL. To check the effective of the protein-DNA interactions, competitive EMSA was performed. Results AP-1-probe was synthesized by homemade annealing buffer synthetic. AP-1 probe bound very efficiently to the AP-1 protein extracted from spinal cord dorsal horn of inflammnatory rats, and the competitive probe did not contain appreciable level of binding ability. Conclusion The optimized non-radioactive EMSA effectively reducing the experimental cost and equipment requirements, and the re- suits are credible and worthy of promotion.