[34] Preparation of cytoplasmic actin

This chapter presents procedure for preparation of cytoplasmic actin. For purification procedure, six to eight brains from 5- to 10-month-old hogs are obtained from a local slaughterhouse within 30 min of the death of the animals. The chopped brains are homogenized in an equal volume of nonpolymerizing extraction buffer. After extraction for a minimum of 30 rain, the homogenate is sedimented by centrifugation at 80,000 g for 90 min. The resulting bright red supernatant is pumped onto a diethylaminoethyl (DEAE)-cellulose column equilibrated with the extraction buffer containing 0.1 M KC1 at a flow rate of approximately 90 ml/hr. Fractions are collected at 14-min intervals and assayed both by electrophoresis in SDS under reducing conditions and by ability to activate the Mg2+-ATPase of rabbit skeletal muscle myosin. The assay is performed by addition of an equal aliquot of each fraction to a constant amount of myosin and measurement of phosphate release.