Purification and Characterization of Tropomyosin Proteins

Tropomyosin proteins were expressed and purified as described previously [6] (link), while poly-histidine tagged proteins were purified on nickel columns (Qiagen) in denaturing conditions (8 M urea, 0.1 M NaH₂PO₄ 0.01 M Tris-Cl). Protein concentrations were determined using 280 nm extinction coefficients of 2,980 cm⁻¹, 27,600 cm⁻¹, 27,550 cm⁻¹ and 64,070 cm⁻¹ for Cdc8, α-SkTm, Tfs1 and Spartin respectively. Protein mass was determined using a Finnegan Mat LCQ ion-trap mass spectroscope. Cosedimentation assays were performed at 25°C as described previously [14] (link). We made use of the fact that acetylated SkTm migrates separately to the unacetylated form on SDS-PAGE to determine the K_D for of the acetylated population of Tm.