Isolation of Mitochondria
Assay of succinate dehydrogenase of after isolation of mitochondria in Cauliflower (Brassica oleracea) using differential centrifugation.
Kelly M. Messick, Rebecca Conner
Department of Biological Sciences, Salisbury University, Salisbury, MD, 21801 U.S.A
Address for correspondence:
Kelly M Messick
Department of Biological Sciences
Salisbury University
Salisbury, MD 21801
Phone: 410-546-2060
Fax: 410-543-6433 e-mail: km96536@gulls.salisbury.edu
Running title: Assay of succinate dehydrogenase.
Introduction
Cell fractionation is a very important procedure in cell biology and can be very useful for studying different organelles. By fractionating, we mean separating or dividing the cell into different component parts. Fractionation results in a series of fractions with only one highly purified organelle or protein present in each one ideally. Fractionation can be done by many different methods including size exclusion chromatography, charge, density, and immunoprecipitation; these are just a few. The two most popular methods are differential centrifugation and density gradient centrifugation because of their flexibility and effectiveness, these methods use gravity to separate organelles based on their size and density(Satori et al 2011). Density gradient centrifugation and differential centrifugation are a lot alike except for the fact that density gradient centrifugation uses a density gradient so the organelles settle at their buoyant densities where as differential centrifugation forms a pellet and supernatant without a density gradient. During differential centrifugation more dense particles pellet before less dense particles so that you are able to separate the original sample into fractions based on density. Fractions are rarely a perfect separation of organelles, normally each fraction will have an organelle that consumes the majority but may be present in other fractions. Because the fractions aren 't a perfect separation of the organelles
Kelly M. Messick, Rebecca Conner
Department of Biological Sciences, Salisbury University, Salisbury, MD, 21801 U.S.A
Address for correspondence:
Kelly M Messick
Department of Biological Sciences
Salisbury University
Salisbury, MD 21801
Phone: 410-546-2060
Fax: 410-543-6433 e-mail: km96536@gulls.salisbury.edu
Running title: Assay of succinate dehydrogenase.
Introduction
Cell fractionation is a very important procedure in cell biology and can be very useful for studying different organelles. By fractionating, we mean separating or dividing the cell into different component parts. Fractionation results in a series of fractions with only one highly purified organelle or protein present in each one ideally. Fractionation can be done by many different methods including size exclusion chromatography, charge, density, and immunoprecipitation; these are just a few. The two most popular methods are differential centrifugation and density gradient centrifugation because of their flexibility and effectiveness, these methods use gravity to separate organelles based on their size and density(Satori et al 2011). Density gradient centrifugation and differential centrifugation are a lot alike except for the fact that density gradient centrifugation uses a density gradient so the organelles settle at their buoyant densities where as differential centrifugation forms a pellet and supernatant without a density gradient. During differential centrifugation more dense particles pellet before less dense particles so that you are able to separate the original sample into fractions based on density. Fractions are rarely a perfect separation of organelles, normally each fraction will have an organelle that consumes the majority but may be present in other fractions. Because the fractions aren 't a perfect separation of the organelles
References: Chen, Q. and E. J. Lesnefsky. 2005. Depletion of cardiolipin and cytochrome c during ischemia increases hydrogen peroxide production from the electron transport chain. Free Radical Biology and Medicine. 40:976-982. Hajek, T., D. Honys, and V. Capkova. 2004. New method if plant mitochondria isolation and sub- fractionation for proteomic assays. Plant Science. 167: 389-395. Satori, C.P., V. Kostal, and E.A. Arriaga. 2011. Individual organelle pH determinations of magnetically enriched endocytic organelles via laser-induced fluorescence detection Williams, E., M. Frana, and L. Erickson. 2012. Cell Fractionation: Assay of Mitochondrial Enzyme Activity. Cell Bio Lab Manuel 2012. 27-29.