Brassica Oleracea Lab Report
Examining the activity rate using DCIP and a spectrophotometer of succinate dehydrogenase isolated from Brassica oleracea mitochondria via mechanical disruption and differential centrifugation
Introduction
Mitochondria are important cellular organelles located in the cytosol of cells and is believed to have originated through an endosymbiotic relationship. The unique double layered membrane structure is responsible for the production of the primary energy currency of the cell; adenosine triphosphate or ATP. Small amounts of ATP that the cell can immediately use as an energy source are available during glycolysis of glucose to pyruvate, but the amount of ATP derived from pyruvate can be greatly increased through further processing. Through reduction-oxidation reactions and the electron …show more content…
An additional 20 mL of cold isolation buffer was added to the paste and ground for an extra 4 minutes. The ground paste was filtered through four layers of cheesecloth into a chilled beaker with 5 mL of cold isolation buffer used to wash the mortar and also poured through the cheesecloth. Any remaining filtrate was squeezed through the cheesecloth to maximise the amount of fraction collected. A 30 mL aliquot of the filtrate was transferred into a chilled 50mL centrifuge tube with the remainder kept on ice. The tube was run at 600 RCF for 10 minutes in a balanced, refrigerated centrifuge set at 4C. The supernatant was Pasteur pipetted into clean, chilled 50mL centrifuge tube and spun at 8500 RCF for 45 minutes at 4C. As the centrifugation stopped, the top 2-5 mL of the supernatant was taken and transferred into a cold a 15mL tube labelled, MFF the rest of the supernatant discarded. The remaining pellet was resuspended with 5 mL of cold assay buffer, and the tube labelled s
Introduction
Mitochondria are important cellular organelles located in the cytosol of cells and is believed to have originated through an endosymbiotic relationship. The unique double layered membrane structure is responsible for the production of the primary energy currency of the cell; adenosine triphosphate or ATP. Small amounts of ATP that the cell can immediately use as an energy source are available during glycolysis of glucose to pyruvate, but the amount of ATP derived from pyruvate can be greatly increased through further processing. Through reduction-oxidation reactions and the electron …show more content…
An additional 20 mL of cold isolation buffer was added to the paste and ground for an extra 4 minutes. The ground paste was filtered through four layers of cheesecloth into a chilled beaker with 5 mL of cold isolation buffer used to wash the mortar and also poured through the cheesecloth. Any remaining filtrate was squeezed through the cheesecloth to maximise the amount of fraction collected. A 30 mL aliquot of the filtrate was transferred into a chilled 50mL centrifuge tube with the remainder kept on ice. The tube was run at 600 RCF for 10 minutes in a balanced, refrigerated centrifuge set at 4C. The supernatant was Pasteur pipetted into clean, chilled 50mL centrifuge tube and spun at 8500 RCF for 45 minutes at 4C. As the centrifugation stopped, the top 2-5 mL of the supernatant was taken and transferred into a cold a 15mL tube labelled, MFF the rest of the supernatant discarded. The remaining pellet was resuspended with 5 mL of cold assay buffer, and the tube labelled s