A Brain Microtubule Protein Preparation Depleted of Mitochondrial and Synaptosomal Components

This chapter discusses a brain microtubule protein preparation depleted of mitochondrial and synaptosomal components. Microtubule protein is isolated by two principal approaches: ion-exchange chromatography on DEAE-Sephadex and a recycling procedure involving warm-induced assembly and cold disassembly with intervening ultracentrifugation steps. Many microtubule purification methods apply one or both of the following methods that are capable of disrupting osmotically fragile organelles: hypotonic extraction of whole brain tissue and/or electromechanical blenders. These extraction methods contribute to destruction of brain mitochondria and synaptosomes, particularly, emulsifying effects by the lipid-rich myelinated white matter in whole brains. The sucrose method for microtubule extraction and isolation minimizes contamination. This chapter outlines a new protocol that minimizes such disruption and contamination while providing excellent yields. Additional information about the molecular characterization of sucrose purified microtubule protein is also presented in the chapter.