The Actin-Toolkit Fluorescence Microscopy was developed to analyze and demonstrate the effect of ligands on actin polymerization, or ligand-induced formation of actin bundles and networks. In the assays, ATTO488-Actin (λex 488nm, λem 523nm) is mixed with the ligand to observe the effects on either G- or F-actin. In contrast to staining with phalloidin conjugates, which can interfere with ligand binding sites on the actin molecule, no such observations were made with ATTO labeled actin. ATTO-Actins are validated to possess the polymerization properties of native actin.
The Toolkit further contains unlabeled actin (0.5mg α-skeletal muscle actin). A mixture of both excludes possible interference of the fluorescent actin conjugates on ligand binding sites. Vinculin tail - a V8 protease cleavage product of vinculin - is an efficient actin bundling vinculin domain, is supplied with the Toolkitas a bundling reference.
Several standardized experiments are described step-by-step in the handbook supplied with the kit. In combination with the actin buffers contained in the kit, error-free handling is guaranteed. The kit contains 4x100µg of α-skeletal muscle ATTO488-Actin conjugates and buffers to keep the actin functional.