membranes membranes are composed of a lipid bilayer and associated proteins ~1/3 of a cell's...
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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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• 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
Membrane Isolation and Characterization
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Cell Disruption Methods
• homogenize• osmotic• presses• N2-cavitation• sonication• freeze-thaw• detergents• chaotropic• enzymatic
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• 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
Membrane Isolation and Characterization
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integral membraneproteins:
soluble in non-ionic detergents(disrupt the lipid bilayer)
peripheral membraneproteins:
soluble in reagents that disruptprotein-protein interactionswithout disrupting the bilayer
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Differential Extraction of Peripheral Membrane Proteins
• low ionic strength at alkaline pH (8-12)
• high salt (> 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 Propertiescritical micellarconcentration
minimum conc. at which micellesform
aggregation #(micellar mol. wt.)
average number of monomers permicelle
critical micellartemp.
minimum temp. at which micellesform
cloud pointtemp. at which micelles formsuper aggregates
• 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
• 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
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Detergent Removal• dialysis
• high cmc, low N• chromatography
• hydrophobic• gel filtration• ion exchange
• replacement• protein precipitation
(eg., PEG)