Travel and Contact  Staff Only  Site Map  Help?     EMBL Hamburg > Courses and Conferences   Introduction   Programme   Sponsors   Speakers   Organizers   Materials (restricted access)   Andy Freer - Dr. Andy Freer, University of Glasgow, Glasgow, G12 8QQ, Scotland, UK. This part of the course will cover many of the aspects encountered when extracting, purifying and crystallizing membrane proteins. The techniques and protocols are mostly analogous to those used for water soluble proteins, except that we introduce a number of additional parameters to account for the introduction of detergent and the presence of lipid. It's a bit like comparing draughts (water) with chess (membrane) - essentially the same game, but with a lot of lateral thinking! We will deal with the concepts and practicalities using detergent to remove the protein from its lipid bilayer, while retaining structural integrity. Which detergent to use and some of the important characteristics of detergents that have to be acknowledged; particularly, critical micelle concentration (CMC). The presence of detergent throughout the purification protocol, and/or the exchange of a detergent that may have been used to solubilise the membrane into a more suitable detergent for purification or crystallization will also be discussed along with basic detergent etiquette. The practical based session should allow students to have hands-on experience in the initial selection of detergent for solubilization and pre-solubilization. We have selected the 3 most popular detergents (LDAO, DDM and BOG) currently in use in membrane investigations. The lecture presentation will highlight the idiosyncrasies of membrane protein extraction, purification and crystallization. This will be illustrated from the experiences gained from working with the myelin proteins and also the light-harvesting and core complexes from photosynthetic bacteria. These examples come from wild type systems where there are several background proteins (both water-soluble and membrane) which have to be removed by employing standard techniques adapted for membrane work. Although the same general rationale that is used for water-soluble protein crystallization is applicable for membrane proteins, several more variables like detergent concentration and type and the use of small amphiphiles is required to aid crystallization. These additional conditions will also be discussed. It is envisaged that both the detergent based lab exercises and Dr Grisshammer's Amidoblack protein assay will give students a good feel for membrane protein work.