ABSTRACT
This chapter discusses the sample preparation and application of different high-performance liquid chromatography (HPLC) methods. The following HPLC methods can be used for preparative and analytical separation of hydrophobic membrane proteins: separation under denaturing conditions and separation under non-denaturing conditions. The proteins from the detergent-poor phase, as well as those from the detergent-rich phase, can be subjected after dilution with cold buffer to further HPLC separation, preparatively and analytically. Although practically all methods for separation of water soluble biopolymers are also used for HPLC separation of hydrophobic proteins, the latter field follows its own rules in some respects. In order to separate membrane proteins, they first have to be solubilized from the membrane structure. The hydrophobic membrane proteins are extracted by the non-ionic detergent Triton X 114. Detergents keep the hydrophobic proteins in solution and prevent their aggregation and unwelcome interaction with the support during HPLC separation.
