Choosing appropriate lysis buffers for protein extraction from acidotic mouse skeletal muscles / 中国组织工程研究

ABSTRACT

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.