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. 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

3. Cell Disruption Methods homogenize osmotic presses N 2 -cavitation sonication freeze-thaw detergents chaotropic enzymatic

4. 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

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

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

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

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

11. 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

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