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.[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.
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