PP06 -...

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EX. 6: Chloroplast Isolation and Separation of Biological Molecules by Chromatography Extraction of membrane-bounded organelles from cells. Extraction of lipid pigments from chloroplasts. Organic separation of non-polar lipids with Thin Layer  Chromatography.  Aqueous separation of colored water-soluble proteins  contained in cell extracts of photosynthetic prokaryotes by  DEAE anion exchange column chromatography.
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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.                                                                          
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Cellular organelles Plant cell Animal cell
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centrioles)
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Plasma membrane : {phospholipid bilayer, integral and peripheral proteins,  cholesterol}
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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)
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Golgi Apparatus: stacked cisternae vesicle lumen
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Mitochondria: two membrane organelle Intermembrane space Outer membrane Inner membrane
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Chloroplast {plastid: family of related  plant organelles}
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Cellular plasma membrane Cell wall 1 µm
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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
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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.
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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.
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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
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Review of 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
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PP06 -...

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