To make a stock concentration of 6.6 μM (20X), reconstitute the lyophilized material in 1.5 ml pure methanol.
Fix cells for 15 minutes using fresh, methanol-free 4% formaldehyde, then rinse three times in PBS for 5 minutes each. Once fixed, please follow CST protocol for immunostaining. Following incubation of the primary and secondary antibodies, Alexa Fluor® 555 Phalloidin can be diluted 1:20 (5 μl per 100 μl assay volume) in PBS and added to the cells. Allow to incubate for 15 minutes at room temperature, then rinse once with PBS. Coverslip slides with ProLong® Gold antifade reagent and examine specimen using appropriate excitation wavelength.
Actin, a ubiquitous eukaryotic protein, is the major component of the cytoskeleton. At least six isoforms are known in mammals. Nonmuscle β- and γ-actin, also known as cytoplasmic actin, are predominantly expressed in nonmuscle cells, controlling cell structure and motility (1). Actin exists mainly as a fibrous polymer, F-actin. In response to cytoskeletal reorganizing signals during processes such as cytokinesis, endocytosis, or stress, cofilin promotes fragmentation and depolymerization of F-actin, resulting in an increase in the monomeric globular form, G-actin (2). Phalloidin is a natually occuring toxic bicyclic peptide found in the deathcap toadstool, Amanita phalloides, that rapidly binds to F-actin with strong affinity (3).
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