Isolation of Mitochondria

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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.
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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 4⁰C. The postnuclear supernatant was removed and centrifuged at 12,000 g for 30 minutes at 4⁰C. 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 MgCl₂, 0.02 M phosphate buffer, pH 7.2). Both samples were stored at
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