Phalloidin Conjugates for Actin Staining

Phalloidin is a bicyclic peptide that belongs to a family of toxins isolated from the deadly Amanita phalloides “death cap” mushroom and is commonly used in imaging applications to selectively label F-actin in fixed cells, permeabilized cells, and cell-free experiments. Phalloidin conjugates have similar affinity for both large and small filaments and bind in a stoichiometric ratio of about one phallotoxin per actin subunit in both muscle and non-muscle cells; they reportedly do not bind to monomeric G-actin.

Fluorescent phalloidin conjugates can be used with other actin-binding proteins such as myosin, troponin, and DNase I. Phalloidin-stained actin filaments are still functional and able to contract. Fluorescent phalloidins can also be used to quantitate the amount of F-actin in cells.

Learn more about additional phalloidin for labeling F-actin

Fluorescent Alexa Fluor and Alexa Fluor Plus dye conjugates of phalloidin are widely used F-actin stains, with color choices across the full spectral range (Figures 1–4). These phalloidin conjugates provide researchers with fluorescent probes that are designed to be superior in brightness and photostability to all other spectrally similar conjugates tested.

Alexa Fluor Plus Phalloidin conjugates have 3–5 times more signal sensitivity and brightness compared to Alexa Fluor conjugates. This extra brightness is especially useful when conducting challenging F-actin imaging, such as with SIM, or STORM, or when reliable staining is needed of stress fibers.

There are other phalloidin conjugates available for staining actin filaments. These conjugates have very little non-specific staining which allows for high-contrast discrimination of actin. Fluorescein and Oregon Green 488 are conjugated to green fluorescent dyes, while BODIPY 558/568 is conjugated to a bright, red fluorescent dye. These phalloidins allow for multiplexing with other fluorescent stains such as fluorescent proteins and Alexa Fluor conjugates including secondary antibodies. Rhodamine Phalloidin is conjugated to the red-orange fluorescent dye, tetramethylrhodamine (TRITC) (Figure 5). Alexa Fluor 546, Alexa Fluor 568, Alexa Fluor 594, and Texas Red-X (Figure 6) phalloidins are available replacements for rhodamine phalloidin since their emission spectra are better separated from green-fluorescent dyes.

Unlabeled phalloidin is also offered for use as a control in blocking F-actin staining by labeled phalloidins and for promoting actin polymerization.