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目的通过引入微量氯化钴作为稳定剂,建立一种更加简便有效提取HIF-1α蛋白的方法。方法取8只体重在22±2克之间雄性8周龄的C57BL/6小鼠,对其左侧下肢行股动脉段离断术制成下肢缺血模型,并分别以右侧下肢为对照;术后1天采用激光多普勒技术检测下肢血流灌注;术后3天取小鼠双侧股四头肌,分别用微量氯化钴法和普通SDS裂解液裂解法来提取总蛋白;用Western Blot对HIF-1α蛋白水平进行检测。结果 8只小鼠手术均成功;术后1天激光多普勒检测结果显示,小鼠缺血下肢血流灌注/健侧肢血流灌注为15.4±4.8%,下肢缺血手术成功;Western Blot显示,使用普通SDS裂解液法,缺血组HIF-1α条带较浅淡,而使用微量氯化钴法,可以清楚地观察到HIF-1α蛋白水平升高。结论微量氯化钴法能够有效稳定HIF-1α蛋白,其提取物用于后续蛋白水平实验显著优于普通的SDS裂解液法。
Objective To establish a simple and effective method for extracting HIF-1α protein by introducing trace cobalt chloride as a stabilizer. Methods Eight male C57BL / 6, 8-week-old male C57BL / 6 mice weighing 22 ± 2 g were used to establish the model of lower extremity ischemia on the left lower extremity. The right lower extremity was used as control. One day after operation, blood flow perfusion of the lower extremities was detected by laser Doppler technique. Bilateral quadriceps muscles of mice were taken 3 days after operation, and total protein was extracted by cobalt chloride method and ordinary SDS lysis solution respectively. Western Blot HIF-1α protein levels were detected. Results All the 8 mice were operated successfully. The results of laser Doppler examination at 1 day after operation showed that blood flow perfusion in the ischemic lower limbs was 15.4 ± 4.8% Showed that the HIF-1α band of the ischemic group was lighter than that of the normal SDS lysis solution. However, the trace amount of HIF-1α protein was clearly observed using the trace cobalt chloride method. Conclusion The method of trace cobalt chloride can effectively stabilize HIF-1α protein, and its extract used in the subsequent protein level experiment is significantly better than the ordinary SDS lysate method.