Preparation of Smooth Muscle α-Actinin

This chapter describes a procedure that is developed specifically for isolation of α-actinin from chicken gizzard in high yield and purity. For preparation of grizzard, it is essential to use chicken gizzards that have been trimmed and stored in 100% glycerol at –20 ° for at least 1 week prior to use. Further steps involves following steps: extraction, which is achieved by dialysis; ammonium sulfate fractionation; anion exchange chromatography; gel filtration. For concentration and storage of α-actinin the protein is precipitated by adding ammonium sulfate to 50% saturation as in step 4. Dissolve the pellet in a buffer of your choice and dialyze extensively to remove ammonium sulfate, α-actinin is stable and soluble in buffers (pH 7.0-8.0) containing almost no salt to as much as 0.6 M KC1. The final solution is make 0.02% in NaN3 and then filtered through a 0.22-p.m Millipore filter. The filtrate collected into sterile tubes and store it at 4 °. α-Actinin in 1 mM KHCO₃ has been stored in this manner for 1 year with no detectable breakdown or loss in its ability to crosslink and to gel actin filaments.