小鼠神经细胞蛋白质的SDS-PAGE聚丙烯酰胺电泳及免疫印记鉴定方法的建立

目的建立一套组合的磷蛋白分析法检测小鼠神经细胞中参与信号转导的蛋白激酶和信号分子。方法培养新生乳小鼠神经细胞，提取蛋白质，并进行SDS-PAGE凝胶电泳，当电泳结束后利用半干式不连续转移缓冲系统将凝腔上的蛋白质转移到PVDF支持膜上，结合免疫印迹和曝光自显影，即得到了小鼠神经细胞中磷蛋白分子（MEK2）的免疫印迹图谱。结果通过上述方法成功地获得了小鼠神经细胞磷蛋白组中磷蛋白分子（MEK2）的免疫印迹图谱。结论利用此组合方法可以检测小鼠神经细胞中参与信号转导的蛋白激酶或信号分子。实验证明本法操作简单，无放射性污染，灵敏度高、特异性强，经济适用，行之有效。

Establishment of a combination method of SDS-PAGE electrophoresis and immunoblotting for identifying neuroproteomics

Objective To identify protein kinases and signaling molecules involved in signaling transduction in mice neurone with a set of cooperated methods of identifying phosphoproteins.Methods The mice neurone were cultured.Then the concentrations of total proteins were assayed after the lysis of neurone.The proteins were separated by SDS-PAGE electrophoresis.Then,the proteins in gels were immediately transfered on PVDF membranes with semi-dry discontinuous electrophesis buffer system.The PVDF membranes were incubated with the specific antibodies of anti-MEK2,dipped in chemiluminescence substrates and put in the block box with X-ray film for autography.The Western blotting maps of MEK2 was obtained.Results The immunoblotting maps of MEK2 were successfully obtained.Conclusion The protein kinases and the signaling molecule involved in the signaling transduction in mice neurone were accurately identified by the method.The experiments prove the methods of identifying phophoproteins are effecient and feasible.