Purification of Titin and Nebulin

This chapter describes two procedures for titin and nebulin purification from rabbit back muscle based on the use of by sodium dodecyl sulfate (SDS). The unusual size of these proteins provides the basis for separation by gel filtration. The first method involves the gel filtration of the entirety of myofibfillar proteins solubilized in SDS. The second method makes use of a novel salt fractionation procedure in which the titin-SDS complex is selectively precipitated from SDS-solubilized myofibrils. This procedure, when combined with gel filtration, provides a rapid and efficient method for titin and nebulin purification. Highly washed rabbit back myofibrils are used as starting material because preparations largely free of endogenous protease can be prepared and stored conveniently. An SDS-polyacrylamide gel electrophoresis system is presented in the chapter that allows the resolution and identification of titin and nebulin.