By Søren B. Nielsen, Daniel E. Otzen (auth.), Jörg H. Kleinschmidt (eds.)
Biological membranes are the fundamental structuring parts of all residing cells. Many enzymatic reactions occur on the membrane-water interface. to achieve particular perception into membrane houses, it truly is for this reason of serious significance to appreciate the complicated nature of the interactions of membrane proteins with lipids. Lipid-Protein Interactions: equipment and Protocols offers a variety of protocols to check protein-lipid interactions, membrane and membrane protein constitution, how membrane proteins have an effect on lipids and the way they're in flip laid low with the lipid bilayer and lipid homes. The equipment defined listed here are all actively used, complementary, and essential to receive finished information regarding membrane constitution and serve as. They contain label-free methods, imaging ideas and spectroscopic methodologies. Written within the winning equipment in Molecular Biology™ sequence structure, chapters contain introductions to their respective issues, lists of the mandatory fabrics and reagents, step by step, without difficulty reproducible protocols, and notes on troubleshooting and averting identified pitfalls.
Authoritative and simply available, Lipid-Protein Interactions: tools and Protocols seeks to serve either specialist and newcomers with its wide selection of the equipment often utilized in this zone of research.
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Additional info for Lipid-Protein Interactions: Methods and Protocols
Example text
Usually micromolar concentrations of proteins should be enough. 3. 1. Preparation of Lipid Vesicles 1. Add 5 mg of desired lipids dissolved in the appropriate organic solvent to a round-bottom flask and dry under vacuum using the rotary evaporator for at least 3 h (see Note 1). 2. Add 1 ml of vesicle buffer and one-third of teaspoon of glass beads. Agitate vigorously on vortex approximately 1 min or 32 V. Hodnik and G. Anderluh until all lipids are removed from the walls of the flask. The temperature of the vesicle buffer should be above the gel–liquid crystal transition temperature.
Notes 1. L1 sensor chip allows capture of liposomes of different composition or size. It is also possible to deposit membrane preparations from cells, such as red blood cell ghosts, plasma membrane remnants, and cellular organelles (5). 2. Small unilamellar vesicles prepared by sonication may also be effectively used. 3. It is important to clean the surface of the sensor chip before the deposition of the liposomes. This is conveniently done by regeneration solutions. 5% SDS or 40 mM octyl glucoside.
Anderluh Fig. 2. HPA and L1 sensor chips offered by Biacore (GE Healthcare). Lipid monolayer, also termed hybrid lipid bilayer, is formed after a solution of small unilamellar vesicles is injected across the surface of the HPA sensor chip. Vesicles then fuse to generate a monolayer supported by a hydrophobic alkane layer. L1 chip possess long aliphatic anchors that efficiently capture intact liposomes. This is the preferred approach for studies of integral membrane proteins and proteins that need both monolayers for efficient membrane binding.