Reconstitution of thin filaments from actin, troponin, and tropomyosin was done by mixing the proteins at a concentration of 2 µM F-actin, 0.5 µM tropomyosin, and 0.48-0.5 µM troponin in F-actin buffer (4 mM imidazole, pH 7.1 at 25C), 2 mM MgCL2, 0.5 mM ATP, 3 mM NaN3, 1 mM DTT). The solution was allowed to incubate overnight before use.
Homsher, E., et al., Calcium regulation of thin filament movement in an in vitro motility assay. Biophys J, 1996. 70(4): p. 1881-92
Thin filaments were then labeled with rhodamine-phalloidin at a 1:1 actin-to-phalloidin ratio in low-salt buffer (in mM: 25 KCl, 25 imidazole, 5 MgCL2, 10 DTT, and 2 EGTA, pH 7.4) and stored overnight a 4°C before use in the in vitro motility assay.
VanBuren, P., et al., Cardiac troponin T isoforms demonstrate similar effects on mechanical performance in a regulated contractile system. Am J Physiol Heart Circ Physiol, 2002. 282(5): p. H1665-71
