Section 11.1 - Probes for Actin

Fluorescently labeled actin (Figure 11.1) is an important tool for investigating cytoskeleton dynamics in vivo.1–5 Molecular Probes offers highly purified actin from rabbit muscle (A-12375), as well as fluorescent actin conjugates labeled with two of our brightest and most photostable dyes. The green-fluorescent Alexa Fluor 488 actin conjugate (A-12373) has excitation and emission maxima similar to fluorescein actin (Figure 7.54), but it is brighter and more photostable, and its spectra are much less pH dependent. The red-orange–fluorescent Alexa Fluor 568 actin conjugate (A-12374, Figure 11.2) is more fluorescent than the spectrally similar Lissamine rhodamine B conjugate. Both of our fluorescent actin conjugates are prepared by reacting amine residues of polymerized F-actin with the succinimidyl ester of the appropriate dye using a modification of the method described by Alberts and co-workers.2 After labeling, the conjugates are subjected to depolymerization and subsequent polymerization to ensure that the actin conjugates are able to assemble properly. The labeled actin that polymerizes is then separated from remaining monomeric actin by centrifugation, depolymerized and packaged in monomeric form.