Lab Report II - BMB 471

Lab Report II - BMB 471 - Analysis of Purification of...

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Analysis of Purification of Cauliflower, Liver, Spinach, and Arabidopsis Tissue BMB 471 - Section 001 Lab co-worker:
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LIST OF ABBREVIATIONS PSII – Photosystem II IU – International Units (micromoles/minute) DCPIP – 2, 6 Dichlorophenolindophenol SDH – Succinate Dehydrogenase BSA – Bovine Serum Albumin HPLC – High Performance Liquid Chromatography FAME – Fatty Acid Methyl Ester TLC – Thin-Layer Chromatography MGDG – Monogalactosyldiacylglycerol DGDG – Digalactosyldiacylglycerol SQDG – sulphoquinovosyldiacylglycerol PG – Phosphotidylglycerol PE – Phosphotidylethanolamine PC – Phosphotidylcholine RESULTS During the first day of this experiment, the fractions FrI, FrSfI, and FrII were successfully produced in spinach leaf chloroplast isolation as illustrated in Figure 1. This isolation procedure allowed us to collect purified chloroplast fractions (FrSfI and FrII) which were then analyzed for PSII * activity. The activity of this marker enzyme is important in that it provides information for determining the purity of each chloroplast-containing fraction produced from isolation; more enzyme activity corresponds to higher concentrations of chloroplast organelles in the fraction. From the assay data illustrated in Figures 2a and 2b, it was observed that the 1:5 diluted fraction FrII caused a reduction in the DCPIP * indicator more quickly than did the fraction FrSf1. Similarly, the total activity of the photosystem in FrII (20.45 IU * ) was calculated to be higher than that of FrSf1 (15.83 IU), resulting in a higher yield for FrII. The yield, or percent total activity, was calculated to be 56.4% for FrII and 43.6% for FrSf1. This makes sense, as pellets (FrII) produced by differential centrifugation contain the bulk of cellular * See list of abbreviations * * 2
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chloroplast compared to the supernatant solution (FrSf1) produced, thus confirming that our chloroplast isolation procedure was successful. Similarly, on the second day of this experiment, we effectively isolated fractions containing mitochondria (FrI, FrII, and FrIII) from cauliflower tissue as illustrated in the isolation flowchart (Figure 3). We confirmed this by performing an assay for SDH * analysis in the cauliflower fraction FrIII. As SDH is a marker enzyme exclusively localized in the mitochondria, the presence and yield (relative to FrI) of these organelles can be determined based on the SDH activity found in the assay. It should be noted that endogenous activity
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This note was uploaded on 03/19/2008 for the course BMB 471 taught by Professor Bowlby during the Spring '08 term at Michigan State University.

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Lab Report II - BMB 471 - Analysis of Purification of...

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