= Solution
\b[A <pyrene-actin assay> can follow assembly continuously.] A small labelled fraction mixed with otherwise unlabelled <actin> gives an increasing signal as the labelled subunits enter <actin filaments>, allowing comparison of lag, growth and plateau. Drug-only and labelled-monomer controls are essential because ToxB might alter <fluorescence> directly; comparable label incorporation and a polymer-mass calibration are needed to compare wild-type and mutant <actin>. An institutional description explains this polymerization-dependent <fluorescence> readout.<Preparation of Pyrenyl-Actin by the Maciver laboratory>[Principle: enhanced fluorescence on polymerization]{ref}
Direct single-filament imaging by <total internal reflection fluorescence microscopy> can separately measure <actin filament> appearance, elongation and shortening, rather than inferring all three from bulk <polymer> mass. It requires matched surface and label conditions, since immobilization can perturb assembly. <light scattering> or <turbidity> can also follow assembly, but aggregates and changes in <actin filament> length or bundling complicate mass estimates. <electron microscopy> can verify <actin filament> morphology and length at selected times, although sampling and preparation make it less convenient for a continuous kinetic curve. Combining a bulk assay with direct <actin filament> imaging addresses different ambiguities.
Back to article page
