微量氯化钴法与普通SDS缓冲液裂解法提取缺氧诱导因子-1α(HIF-1α)蛋白效率的比较

Comparison the Efficiency of Extracting Hypoxia-inducible Factor-1α(HIF-1α) Protein by Cobalt Chloride Included Buffer and Ordinary SDS Buffer

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 protein extraction method of HIF-1α extraction via taking cobalt chloride as a stabilizer. Methods Eight 8-week-old male C57BL / 6 mice with body weight at 22 1±~ 2 g were used to perform the limb ischemia models. The left femoral artery segment of each mouse was ligated, and the right limb was taken as control. Laser Doppler detection was employed to detect limb perfusion in both sides, and HIF-1α was detected by Western Blot. Results Limb ischemia model was successful, because their left limb has only 15.4±4.8% perfusion of the right. Western Blot showed that HIF-1α was almost undetectable in non-ischemie limbs and shallow in isehemia limbs if SDS buffer was used ; while HIF-1α were observed clearly if cobalt was added. Conclusion Cobalt chloride RIPA buffer was better than ordinary SDS buffer to extract and detect HIF-1α by stabilizing HIF-1α.