Alterations of myosin isoenzymes and ADP/ATP carrier in Goldblatt hypertensive rats (part 4)

Mitochondrial membrane protein extraction and determination of AAC content: The left ventricles were minced, homogenized and centrifuged, and the mitochondrial membranes were separated according to the method of Tzagoloff et al . The minced left ventricular myocardium was homogenized in 50 mmol Tris-HCl buffer (pH 7.4) containing 250 mmol sucrose and 1 mmol EDTA with a glass homogen-izer at 4°C. All the following procedures were performed at 4°C. The homogenate was centrifuged at 1000 g for 10 mins. The supernatant was then centrifuged at 8000 g for 20 mins. The pellet was suspended in 50 mmol Tris-HCl buffer (pH 7.4) containing 250 mmol sucrose and centrifuged at 8000 g for 20 mins. The pellet was resuspended in the same solution.The extracts were treated with 20 mmol Tris-HCl buffer (pH 6.8), 2% SDS, 2% 2-mercaptoethanol and 40% glycerol, and boiled for 5 mins. The treated mitochondrial membrane extracts were placed directly on SDS 12.5% polyacrylamide slab gel (NPU-12.5L, PG-R ATTO Co, Tokyo, Japan). The electrophoresis buffer contained Tris 0.3%, glycine 1.44% and SDS 0.1%. Electrophoresis was carried out for 2 h with constant electrical current of 20 mA at room temperature. The gels were stained with Coomassie brilliant blue and were destained with 7.5% acetic acid and 5% methanol. Bands containing the AAC component revealed on the gel were measured by densitometer . All values are expressed as means ± SEM, tested statistically significant differences between Goldblatt and control rats, Student’s t test was used to test differences between Goldblatt and control rats. You now have a chance to make sure your disease is under complete control. Being able to buy your drugs from my canadian pharmacy means you will be spending less time and money every time, getting the same high quality.

This entry was posted in Hypertrophy and tagged ADP/ATP carrier, Goldblatt hypertensive rat, Hypertrophy, Myosin isoenzyme, Pyrophosphate gel electrophoresis.