6 - EX 6 Chloroplast Isolation and Separation of Biological Molecules by Chromatography Extraction of membrane-bounded organelles from cells

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EX. 6: Chloroplast Isolation and Separation of Biological Molecules by Chromatography • Extraction of membrane-bounded organelles from cells. • Extraction of lipids from photosynthetic tissue and separate them with TLC. • Separation of colored water-soluble proteins contained in cell extracts of photosynthetic prokaryotes. Membrane-bounded organelles? • Usually structures confined to eukaryotic cells. • Made up of a lumen (space) filled with fluid. • Materials of lumen kept separate from cytoplasm by membranes. Cellular organelles Plant cell Animal cell Inclusions: non-membrane-bounded Plasma membrane: {phospholipid bilayer, integral and peripheral proteins, cholesterol} Vesicles: single membrane surrounding a lumen {e.g., lysosomes and microbodies. . . 0.5-1.0 μm}
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Cisternal space ER membrane Ribosomes (inclusions) Golgi Apparatus: stacked cisternae vesicle lumen Mitochondria: two membrane organelle Intermembrane space Outer membrane Inner membrane Chloroplast {plastid: family of related plant organelles} Cellular plasma membrane Cell wall 1 μm stroma Granum {stack of thylakoids} Thylakoid
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Which of the following is not a membrane-bound organelle? • A. Chloroplast • B. Ribosome • C. Mitochondion • D. Golgi body Strategy for isolating cellular organelles • 1. Select appropriate source of cells. • 2. Place intact cells in a protected environment. • 3. Break open cells. • 4. Separate organelle of interest from other components. 1. Select an appropriate source of cells. For chloroplasts, choose green leaves. • Choose a plant from which it is easy to extract intact organelles. • Discard, where feasible, plant parts that don’t contain chloroplasts. 2. Place cells in a protected environment • A) Osmotic support : surround cell with solution that provides an osmolarity as large or larger than the osmolarity of the lumen [0.40 M sucrose or 0.25 M NaCl]. • Avoid disrupting organelle membranes by avoiding hypotonic solutions Osmolarity • Solute concentration expressed as molarity • For non-dissociating molecules, osmolarity roughly equal to molar concentration – e.g., 0.11 M sucrose solution = osmolarity of 0.10 • Osmolarity is approximately equal to the sum of the osmolarity of each kind of particle in solution • Osmolarity within each organelle about equal to that of cytoplasmic matrix Osmosis terms movement of water across a selectively permeable membrane ISOTONIC: SAME SOLUTE CONCENTRATION HYPERTONIC: HIGH SOLUTE CONCENTRATION HYPOTONIC: LOW SOLUTE CONCENTRATION
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Isotonic: [solute] equal inside and outside cell, no net flow of water out or in cell/organelle Hypertonic: [solute] outside membrane greater than inside, net outflow of water, cell/organel e becomes flaccid Hypotonic: [solute] outside membrane greater than inside, net inflow of water, cell/organelle bursts! Osmosis:movement of water across a selectively permeable membrane
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This note was uploaded on 02/22/2010 for the course BIO 50100 taught by Professor Brand during the Fall '09 term at University of Texas at Austin.

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6 - EX 6 Chloroplast Isolation and Separation of Biological Molecules by Chromatography Extraction of membrane-bounded organelles from cells

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