LDH isozymes electrophoresis

The skeletal muscle samples of pikas were homogenized on ice as a 1:4 (W/V) dilution in 0.9% physiological saline. The homogenate was centrifuged at 15,000 r/min at 4°C for 10 min and the supernatant collected. LDH isozymes electrophoresis was performed with a DY-200 steady current and voltage electrophoresis apparatus (Beijing Liuyi Instrument Factory, Beijing, China). The electrode buffer was Tris-glycine (pH 8.3), and 6 μl of the samples were loaded. The current was 10 mA in the stacking gel and then 25 mA in the separating gel. The LDH bands were stained in the dark at 37°C in a mixture of 4 ml of 5 mg/ml NAD⁺, 2.5 ml of 0.1 mol NaCl, 10 ml of 1 mg/ml nitrobenzene thiocyanate chloride (NBT), 1 ml of 1 mg/ml phenazine methosulfate (PMS), 2.5 ml of 1 mol/l sodium lactate and 0.5 mol/l phosphate buffer (pH 7.5) for 30 min. After rinsing the gels with distilled water, they were stored in 10% glycerol and 7% acetic acid.