Isolation of alpha-connectin, an elastic protein, from rabbit skeletal muscle

alpha-Connectin (also called titin 1) has been isolated from rabbit back muscle. Myofibrils were well washed with 5 mM NaHCO3 and then extracted with 0.2 M sodium phosphate, pH 7.0. The extract was dialyzed against 0.1 M potassium phosphate, pH 7.0, to sediment myosin. The supernatant, adjusted to 0.18 M potassium phosphate, pH 7.0, and 4 M urea, was subjected to DEAE Toyopearl column chromatography. beta-Connectin was eluted in the flow-through fraction and alpha-connectin was eluted at around 0.1 M NaCl, when a 0 to 0.25 M NaCl gradient was applied. The separated alpha-connectin was dialyzed against 0.2 M potassium phosphate, pH 7.0. The resultant alpha-connectin showed the same mobility as that in an SDS extract of rabbit back muscle on SDS gel electrophoresis using 1.8% polyacrylamide gels. A monoclonal antibody against chicken breast muscle beta-connectin reacted with the alpha-connectin isolated from rabbit back muscle.