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

• 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

Cell Disruption Methods

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

• 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

integral membraneproteins:

soluble in non-ionic detergents(disrupt the lipid bilayer)

peripheral membraneproteins:

soluble in reagents that disruptprotein-protein interactionswithout disrupting the bilayer

Differential Extraction of Peripheral Membrane Proteins

• low ionic strength at alkaline pH (8-12)

• high salt (> 0.15 M)• chaotropic salts, urea

Detergents• amphiphilic compounds

• hydrophobic and hydrophilic moieties

• surface activity• soluble micelles

Detergent Structure

• Polar Groups• ionic

• anionic• cationic• zwitterionic

• non-ionic• polyoxyethyl• glucosides

• Hydrophobic Groups• alkyl chains

branching phenyl, etc.

• steroid skeleton

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

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

Detergent Removal• dialysis

• high cmc, low N• chromatography

• hydrophobic• gel filtration• ion exchange

• replacement• protein precipitation

(eg., PEG)