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Membranes

Membranes. membranes are composed of a lipid bilayer and associated proteins ~1/3 of a cell's proteins are directly associated with membranes many soluble proteins are contained within membrane bound organelles. Membrane Isolation and Characterization. selection of tissue

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Membranes

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  1. Membranes • membranes are composed of a lipid bilayer and associated proteins • ~1/3 of a cell's proteins are directly associated with membranes • many soluble proteins are contained within membrane bound organelles

  2. Membrane Isolation and Characterization • selection of tissue • selection of membrane fraction • cell disruption conditions • preparation of membranes • differential centrifugation • density gradient centrifugation • solubilization of membrane proteins • analysis and isolation of proteins

  3. Cell Disruption Methods • homogenize • osmotic • presses • N2-cavitation • sonication • freeze-thaw • detergents • chaotropic • enzymatic

  4. Membrane Isolation and Characterization • selection of tissue • selection of membrane fraction • cell disruption conditions • preparation of membranes • differential centrifugation • density gradient centrifugation • solubilization of membrane proteins • analysis and isolation of proteins

  5. Differential Extraction of Peripheral Membrane Proteins • low ionic strength at alkaline pH (8-12) • high salt (> 0.15 M) • chaotropic salts, urea

  6. Detergents • amphiphilic compounds • hydrophobic and hydrophilic moieties • surface activity • soluble micelles

  7. Detergent Structure • Polar Groups • ionic • anionic • cationic • zwitterionic • non-ionic • polyoxyethyl • glucosides • Hydrophobic Groups • alkyl chains •  branching •  phenyl, etc. • steroid skeleton

  8. Detergent Properties • cmc and N will be affected by temp., pH, ionic strength • generally cmc and N inversely related

  9. Choosing a Detergent • efficiency of solubilization • protein:detergent ratio • lipid:detergent ratio • effect on protein structure • effect on protein activity • effect on protein assay • effect on separation technique • ease of removal

  10. Detergent Removal • dialysis • high cmc, low N • chromatography • hydrophobic • gel filtration • ion exchange • replacement • protein precipitation (eg., PEG)

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