Membranes

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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

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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

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Cell Disruption Methods

• homogenize
• osmotic
• presses
• N2-cavitation
• sonication
• freeze-thaw
• detergents
• chaotropic
• enzymatic

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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

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Differential Extraction of Peripheral Membrane
Proteins

• low ionic strength at alkaline pH (8-12)
• high salt (gt 0.15 M)
• chaotropic salts, urea

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Detergents

• amphiphilic compounds
• hydrophobic and hydrophilic moieties

• surface activity
• soluble micelles

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Detergent Structure

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

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Detergent Properties

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

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Choosing a Detergent

• efficiency of solubilization
• proteindetergent ratio
• lipiddetergent ratio
• effect on protein structure
• effect on protein activity
• effect on protein assay
• effect on separation technique
• ease of removal

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Detergent Removal

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