Biology Reference
In-Depth Information
CHAPTER
22
Identification of
Multimolecular Complexes
and Supercomplexes in
Compartment-Selective
Membrane Microdomains
Panagiotis Mitsopoulos and Joaqu´n Madrenas
Department of Microbiology and Immunology, McGill University, Montreal, QC, Canada
CHAPTER OUTLINE
Introduction and Rationale....................................................................................... 413
22.1 Detergent-Insoluble Glycosphingolipid (DIG) Microdomain Isolation ...................415
22.1.1 Materials ................................................................................... 415
22.1.1.1 Reagents ........................................................................... 415
22.1.1.2 Equipment......................................................................... 415
22.1.1.3 Buffers and Sucrose .......................................................... 415
22.1.2 Method...................................................................................... 417
22.1.2.1 Isolation of DIG Fractions ................................................... 417
22.1.2.2 Immunoprecipitation of DIG Fractions ................................ 417
22.2 Plasma Membrane Isolation.............................................................................418
22.2.1 Materials ................................................................................... 418
22.2.1.1 Reagents ........................................................................... 418
22.2.1.2 Buffers and Solutions......................................................... 418
22.2.1.3 Equipment......................................................................... 419
22.2.2 Method...................................................................................... 420
22.2.2.1 Silica Coating..................................................................... 420
22.2.2.2 Fractionation ..................................................................... 420
22.3 Cardiolipin-Enriched Mitochondrial Membrane Microdomain Isolation ...............421
22.3.1 Materials ................................................................................... 421
22.3.1.1 Reagents ........................................................................... 421
22.3.1.2 Buffers .............................................................................. 421
22.3.2 Method...................................................................................... 422
22.3.2.1 Mitochondria Isolation........................................................ 422
22.3.2.2 Cardiolipin-Enriched Microdomain Isolation ........................ 422
