Isolation of Mitochondria - 2221 Words

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.†¦show more content†¦A fraction that has high content of the specific organelle and low contamination by other organelles is desired however a fraction with highly purified mitochondria is better even if the most mitochondria is found in other fractions. The purity of mitochondrial fractions is usually determined by enzyme marker detection assay(Hajek et al 2004). In our experiment we will use differential centrifugation to isolate the mitochondria of cauliflower and then assay SDH activity using a fixed time assay. We will then measure protein content in our fractions and calculate specific activity and total activity of our fractions. Materials and Methods Mitochondrial Isolating We used to florets from the cauliflower and disrupted the cell walls using a cold isolation buffer (0.3 M D-mannitol, 0.02 M phosphate buffer, pH 7.2) and an abrasive, we then strained the homogenate with cheesecloth into a centrifuge tube suppoerted by ice. The homogenate was then centrifuged at 600 g for 10 minutes at 4⠁ °C. The postnuclear supernatant was removed and centrifuged at 12,000 g for 30 minutes at 4⠁ °C. The post mitochondrial supernatant was removed and the mitochondrial pellet was resuspended in a cold assay buffer (0.3 M D-mannitol, 0.01 M KCl, 0.005 M MgClâ‚‚, 0.02 M phosphate buffer, pH 7.2). Both samples were stored atShow MoreRelatedSummary Of Mut-OX In A549 Cells1163 Words   |  5 PagesIn this SA, we will study translocation of DNA damage repair proteins from the cytoplasm to mitochondria in response to mtDNA damage induced by mt-OX in A549 cells. We will treat A549 cells with mt-OX as mentioned in SA#...., followed by using a SILAC method as desc ribed above. We will perform differential ultracentrifugation to isolate cellular fractions as previously described (18). The purity of cytoplasmic and mitochondrial fractions will be assessed by western blotting using IÃŽ ºB-ÃŽ ± and Tom20Read MoreEssay On Lysosome869 Words   |  4 Pagesto the autophagosome membrane and become the active form needed during the autophagic process, LC3BII (Pankiv, 2007; Kabeya, 2000). 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