Isolation of a membrane protein by chromatofocusing: cytochrome b-561 of the adrenal chromaffin granule.
Chromatofocusing, a form of ion-exchange chromatography in which proteins are separated on the basis of their differing isoelectric points, has been adapted for use with membrane proteins, solubilized by the non-ionic detergent Nonidet P-40. Using a two-step detergent extraction followed by chromatofocusing under high pressure, the highly hydrophobic protein cytochrome b-561 was isolated from chromaffin granule membranes and purified to near homogeneity in a functionally active form, in less than 5 h. Chromatofocusing conditions were optimized empirically since the behaviour of the chromaffin granule membrane proteins conformed less to the theory than that of soluble proteins, and the various factors affecting yield and resolution are discussed. The speed, high resolution and focusing effect could make this method particularly suitable for rapid isolation in a functionally active form of the many membrane proteins that are unstable in dilute solution and when removed from their lipid environment.
D002837:Chromaffin Granules / Q000032:analysis*; D002838:Chromaffin System / Q000032:analysis*; D002852:Chromatography, Ion Exchange / Q000379:methods*; D003573:Cytochrome b Group / Q000302:isolation & purification*; D003902:Detergents; D007525:Isoelectric Focusing; D008565:Membrane Proteins / Q000302:isolation & purification*; D017830:Octoxynol; D011092:Polyethylene Glycols
D003573:Cytochrome b Group; D003902:Detergents; D008565:Membrane Proteins; C023646:cytochrome b561; D011092:Polyethylene Glycols; D017830:Octoxynol; C010615:Nonidet P-40