筛选适合酸中毒小鼠骨骼肌组织蛋白提取的裂解液

Choosing appropriate lysis buffers for protein extraction from acidotic mouse skeletal muscles

背景:RIPA Buffer对不同组织细胞总蛋白的提取效率有差异,并不通适于所有的组织样本。目的:筛选适合酸中毒小鼠骨骼肌组织蛋白提取的裂解液,为研究骨骼肌萎缩奠定基础。方法:3月龄健康雄性C57BL/6小鼠20只,体质量25-30 g,由山西医科大学实验动物中心提供。麻醉后脱臼处死小鼠,分离获取下肢腓肠肌。实验分为2组:HCl诱导酸中毒组每日定时供给10 g混合10 mL 0.4 mol/L HCl的饲料;对照组饲料中混合同等量的H2O,连续7 d。比较RIPA Buffer、Original Buffer和JP Buffer对酸中毒小鼠骨骼肌组织蛋白的提取效果;蛋白免疫印迹实验检测AKT、p-AKT(Thr308)、rp S6和p-rp S6(Ser235/236)的表达;RT-qPCR定量分析GLUT4的表达。结果与结论:①3种裂解液对骨骼肌组织蛋白的提取能力有差异,JP Buffer提取蛋白产量高,但是目的蛋白信号并不强;RIPA Buffer提取蛋白产量相对偏低;Original Buffer提取足量骨骼肌组织蛋白,蛋白免疫印迹显示目的蛋白条带清晰;②蛋白免疫印迹实验评分显示,Original Buffer总分高于其他2种裂解液;③蛋白免疫印迹实验结果显示,酸中毒组和对照组AKT、rpS6磷酸化程度均无变化;④RT-qPCR定量分析显示,酸中毒组和对照组RNA水平GLUT4表达无变化; ⑤结果提示,OriginalBuffer是提取骨骼肌组织蛋白的最佳裂解液;短期HCl诱导酸中毒组AKT信号通路未激活,延长酸中毒时间是否会激活该信号通路值得研究。针对不同实验样本选择合适的裂解液是保证蛋白免疫印迹实验结果可信度的前提。

BACKGROUND: RIPA Buffer exhibits different extraction efficiencies of proteins of cells and tissues, which is not appropriate for all samples. OBJECTIVE: To achieve an optimal lysis buffer for skeletal muscle protein extraction in mice of acidosis, and to provide basis for studies on skeletal muscle atrophy. METHEDS: Twenty male healthy C57BL/6 mice, aged 3 months, weighting 25-30 g, were provided by Laboratory Animal Center of Shanxi Medical University. The mice were sacrificed after anesthesia, and the gastrocnemius muscle of lower extremity was isolated. There were two groups: acidosis group was given 10 g of feed mixed with 0.4 mol/L hydrochloric acid (10 mL), and control group received 10 g of feed mixed with same volume of water, for 7 consecutive days. The effect of RIPA Buffer, Original Buffer and JP Buffer on the skeletal muscle protein extraction in mice of acidosis was compared. The expression levels of AKT, p-AKT (Thr308), rpS6 and p-rpS6 (Ser235/236) were detected by western blot assay. GLUT4 mRNA expression was examined by RT-qPCR. RESULTS AND CONCLUSION:(1) Different buffers generated different protein-yields. The protein yield was highest in JP Buffer, but the target protein signal was not high. The protein yield was low in RIPA Buffer. Original Buffer could extract sufficient proteins, and had clear band detected by western blot assay.(2) Western blot assay scores in Original Buffer were higher than those of other two buffers.(3) Western blot assay results showed that the extent of phosphorylation in both groups showed no significant changes.(4) GLUT4 mRNA expression level examined by RT-qPCR showed no significant changes in both groups.(5) These results indicate that Original Buffer is optimal lysate of skeletal muscle protein extraction. Inactivated AKT signaling pathway is seen in the short-term hydrochloric acid-induced acidosis group, so whether lengthening acidosis time can activate the signaling pathway. Selecting the optimal lysis buffer for different samples is premise to ensure western blot assay results.