There are a variety of fluorescent and biotinylated phalloidins that label F-actin. These conjugates are water-soluble, allowing for convenient labeling, identifying, and quantitating of F-actin in tissue sections, cell cultures, and cell-free experiments. The binding properties of phalloidin conjugates do not change appreciably with actin from different species, including plants and animals. In addition, they exhibit negligible non-specific staining, resulting in a clear and distinct contrast between stained and unstained areas.
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.
|Probe||Excitation (nm)||Emission (nm)||Cat. No.|
|Alexa Fluor 350 Phalloidin||346||442||A22281|
|Alexa Fluor 488 Phalloidin||496||518||A12379|
|Alexa Fluor 532 Phalloidin||531||554||A22282|
|Alexa Fluor 546 Phalloidin||556||570||A22283|
|Alexa Fluor 555 Phalloidin||555||565||A34055|
|Alexa Fluor 568 Phalloidin||578||600||A12380|
|Alexa Fluor 594 Phalloidin||581||609||A12381|
|Alexa Fluor 633 Phalloidin||632||647||A22284|
|Alexa Fluor 635 Phalloidin||633||647||A34054|
|Alexa Fluor 647 Phalloidin||650||668||A22287|
|Alexa Fluor 660 Phalloidin||663||690||A22285|
|Alexa Fluor 680 Phalloidin||679||702||A22286|
|Alexa Fluor Plus|
|Alexa Fluor Plus 405 Phalloidin||405||450||A30104|
|Alexa Fluor Plus 555 Phalloidin||555||565||A30106|
|Alexa Fluor Plus 647 Phalloidin||650||668||A30107|
|Alexa Fluor Plus 750 Phalloidin||758||784||A30105|
|Phalloidin eFluor 520||488||520||59-6559-05|
|Phalloidin eFluor 570||555||570||41-6559-05|
|Phalloidin eFluor 660||633||647||50-6559-05|
|Other phalloidin conjugates|
|BODIPY 558/568 Phalloidin||558||569||B3475|
|Oregon Green 488 Phalloidin||496||520||O7466|
|Texas Red-X Phalloidin||591||608||T7471|
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.
Figure 2. U2OS cells labeled with Alexa Fluor Plus 647 Phalloidin. U2OS cells labeled using NucBlue Live ReadyProbes Reagent, CellLight Talin-GFP BacMam 2.0, CellLight Mitochondria-RFP BacMam 2.0, and Alexa Fluor Plus 647 Phalloidin show multiplexing capability and staining specificity. Cells were mounted in ProLong Glass Antifade Mountant. Images were generated using an EVOS M7000 Imaging System with an Olympus 60X Apochromat Oil objective using DAPI, GFP, RFP, and Cy5 EVOS light cubes.
Figure 3. HeLa cells labeled with Alexa Fluor Plus 750 Phalloidin. HeLa cells labeled using NucBlue Live ReadyProbes Reagent, CellLight Talin-GFP BacMam 2.0, CellLight Mitochondria-RFP BacMam 2.0, and Alexa Fluor Plus 750 Phalloidin show multiplexing capability and staining specificity. Cells were mounted in ProLong Glass Antifade Mountant. Images were generated using an EVOS FL Auto 2 Imaging System with an Olympus 60X Apochromat Oil objective using DAPI, GFP, RFP, and Cy7 EVOS light cubes.
Figure 4. HeLa cells labeled with Alexa Fluor Plus 405 Phalloidin. HeLa cells labeled using Alexa Fluor Plus 405 Phalloidin, CellLight Talin-GFP BacMam 2.0, CellLight Mitochondria-RFP BacMam 2.0, and DRAQ5 show multiplexing capability and staining specificity. Cells were mounted in ProLong Glass Antifade Mountant. Images were generated using an EVOS FL Auto 2 Imaging System with an Olympus 60X Apochromat Oil objective using DAPI, GFP, RFP, and Cy5 EVOS light cubes.
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.
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