An Experimental and Statistical Analysis of Sdh Activity as Compared in Liver, Kidney, and Heart Homogenates of the Bos Taurus
Jeffrey Shenfeld
An Experimental and Statistical Analysis of SDH activity as compared in Liver, Kidney, and Heart Homogenates of the Bos taurus
Methods
The three tissues being analyzed in this experiment, those of the kidney, heart and liver, were taken from the animal Bos taurus. The tissue homogenates used were made by adding 1 gram of tissue to 20 ml of sucrose phosphate buffer. The buffer was composed of 250 mM sucrose, 50 mM NaPO4, with a pH of 7.4. This mixture was homogenized with a high-speed blender for 3 pulses for a duration of 10 seconds each. The homogenates were then filtered through a cheesecloth and stored at -70° C.
The homogenate used in this experiment was liver homogenate. Five different samples and a blank were made in order to test SDH activity in the three homogenates. The samples were prepared using varying amounts of 50 mM NaPO4 pH 7.4, which was added to all samples. 3000 μl of solution for the assay was prepared with final concentrations of 83.3 μM of 2,6-dichloroindophenol (DCIP), and 10 mM succinate in 50 mM NaPO4 pH 7.4 The final concentrations of malonate used were: 1mM, 2mM, and 3mM and were used in tubes 3-6. SDH activity was measured against malonate activity, since malonate was expected to inhibit SDH activity. The control used in this assay was used to determine the background amount of SDH activity, using the same setup bit no succinate in 50 mM NaPO4 pH 7.4 or malonate was added to the control. After the samples were prepared, 250 μl of homogenate was added to each tube successively.
Homogenate was added right before analysis in spectrophotometer. Samples were analyzed via spectrophotometer at 600 nm, and the spectrophotometer was set to 1.00. The tube without malonate was used as a blank.
Reactions were then incubated at 30°C for 10 minutes, and then placed on ice for 5 minutes. After incubation and cooling, the absorbances of the various samples were analyzed via spectrophotometry, and the final
An Experimental and Statistical Analysis of SDH activity as compared in Liver, Kidney, and Heart Homogenates of the Bos taurus
Methods
The three tissues being analyzed in this experiment, those of the kidney, heart and liver, were taken from the animal Bos taurus. The tissue homogenates used were made by adding 1 gram of tissue to 20 ml of sucrose phosphate buffer. The buffer was composed of 250 mM sucrose, 50 mM NaPO4, with a pH of 7.4. This mixture was homogenized with a high-speed blender for 3 pulses for a duration of 10 seconds each. The homogenates were then filtered through a cheesecloth and stored at -70° C.
The homogenate used in this experiment was liver homogenate. Five different samples and a blank were made in order to test SDH activity in the three homogenates. The samples were prepared using varying amounts of 50 mM NaPO4 pH 7.4, which was added to all samples. 3000 μl of solution for the assay was prepared with final concentrations of 83.3 μM of 2,6-dichloroindophenol (DCIP), and 10 mM succinate in 50 mM NaPO4 pH 7.4 The final concentrations of malonate used were: 1mM, 2mM, and 3mM and were used in tubes 3-6. SDH activity was measured against malonate activity, since malonate was expected to inhibit SDH activity. The control used in this assay was used to determine the background amount of SDH activity, using the same setup bit no succinate in 50 mM NaPO4 pH 7.4 or malonate was added to the control. After the samples were prepared, 250 μl of homogenate was added to each tube successively.
Homogenate was added right before analysis in spectrophotometer. Samples were analyzed via spectrophotometer at 600 nm, and the spectrophotometer was set to 1.00. The tube without malonate was used as a blank.
Reactions were then incubated at 30°C for 10 minutes, and then placed on ice for 5 minutes. After incubation and cooling, the absorbances of the various samples were analyzed via spectrophotometry, and the final
References: Clendening, B., St. Angelo C.J., Krause M.K., Diangelo J.R., Vallier L.G. 2013. Introductory Cell Biology and Genetics, Bio 11 Laboratory Manual. Hofstra University, NY. DuBois, Kenneth P. et al. "Studies on the Intermediary Carbohydrate Metabolism of Aquatic Animals." Department of Pharmacology, University of Chicago, Chicago, and the Marine Studies Biological Station, Marineland, Florida, (1947): n. pag. Print. LEISCHER, S ., G. ROUSER, B. FLEISCHER, A. CASU, and G. KRITCHEVSKY. 1967 . Lipid composition of mitochondria from bovine heart, liver, and kidney. J. Lipid Res. 8:170. Knisely, Karin. A Student Handbook for Writing in Biology. Sunderland, MA: Sinauer, 2009. Print.