Phalloidin Conjugates
Phalloidin is a bicyclic heptapeptide isolated from the poisonous death cap mushroom, Amanita phalloides. It possesses a high binding affinity for the grooves between F-actin subunits over monomeric G-actin. Compared to actin antibodies, non-specific binding of phalloidin is negligible, thus providing minimal background and high-contrast during cellular imaging. Once bound to F-actin, phalloidin shifts the equilibrium of monomers and filaments toward the filaments position, and inhibits ATP-hydrolysis. The interaction stabilizes actin filaments by preventing subunit dissociation, and it promotes actin polymerization by lowering the critical concentration.
When conjugated to fluorescent dyes, Phalloidin can be used at nanomolar concentrations to label and visualize filamentous actin in fixed and permeabilized cells, cell cultures and cell-free experiments, as well as, formaldehyde-fixed and permeabilized tissue sections. Phalloidin conjugates exhibit similar affinity for all types and sizes of actin filaments, binding in a stoichiometric ratio of 1:1 (phallotoxin : actin) in both muscle and nonmuscle cells. In comparison to antibodies, phalloidin derivatives' are small (< 2 kDa) and phalloidin bound filaments do not impede functional properties of the filaments. The small size also permits for denser F-actin labeling producing more detailed stains when imaged at higher resolutions. In addition, because actin is evolutionarily conserved, the binding properties of phalloidin derivatives can be utilized in staining a wide range of animal and plant cells.
Available Phalloidin Conjugates for Staining F-actin
Table of Contents
Phalloidin iFluor™ Conjugates
Exploiting the excellent fluorescence properties of our iFluor™ dyes, AAT Bioquest has developed a series of phalloidin conjugates spanning the full color spectrum which selectively bind to F-actins. Used at nanomolar concentration, these iFluor™ phalloidin derivatives are convenient for many actin-related assays. Such assays include labeling, identifying, and quantitating F-actins in formaldehyde-fixed and permeabilized tissue sections, cell-cultures, or cell-free experiments. Compared to traditional fluorescein isothiocynanate (FITC) and rhodamine conjugates, Phalloidin-iFluor™ conjugates offer F-actin stains that are superior in brightness and photostability. Phalloidin-iFluor™ conjugates are equivalent, and in most cases superior, to Alexa Fluor® conjugates in performance.
We encourage the research community to try our Phalloidin-iFluor™ conjugates with their respective protocols for a more intense fluorescence in their F-actin staining. Phalloidin-iFluor™ 700, iFluor™-750 and iFluor™-790 conjugates are among the few near-infrared stains available with sufficient spectral separation from commonly used red fluorophores.
Table 1. Overview of fluorescent phalloidin conjugates
| Specification ▲ ▼ | Value ▲ ▼ |
| Application | Fluorescent imaging of filamentous actin (F-actin) |
| Sample type | Fixed & permeabilized cells, cell cultures, cell-free experiments, formaldehyde-fixed & permeabilized tissue sections |
| Cellular localizatoin | F-actin of the cytoskeleton and contractile apparatus of muscle cells (does not bind to monomeric G-actin) |
| Live/Fixed Cells | Fixed cells only |
| Permeability | Cell impermeant |
| Multiplexing | Yes |
| Platform | Fluorescence microscope and imaging platforms |
Table 2. Phalloidin iFluor™ Conjugates
| Conjugate ▲ ▼ | Mol. Wt. ▲ ▼ | Ex/Em (nm) ▲ ▼ | Filter Set (nm) ▲ ▼ | ε ▲ ▼ | Φ ▲ ▼ | Unit Size ▲ ▼ | Price ▲ ▼ |
| Phalloidin-iFluor™ 350 | ∼1300 | 344/448 | DAPI | 20,000 | 0.95 | 300 Tests | $153 |
| Phalloidin-iFluor™ 405 | ∼1400 | 402/425 | DAPI | 37,000 | 0.91 | 300 Tests | $153 |
| Phalloidin-iFluor™ 488 | ∼1900 | 491/516 | FITC | 75,000 | 0.9 | 300 Tests | $153 |
| Phalloidin-iFluor™ 514 | ∼1800 | 527/554 | TRITC | 80,000 | 0.83 | 300 Tests | $153 |
| Phalloidin-iFluor™ 532 | ∼1800 | 543/563 | TRITC | 90,000 | 0.68 | 300 Tests | $153 |
| Phalloidin-iFluor™ 555 | ∼1300 | 556/569 | TRITC | 100,000 | 0.64 | 300 Tests | $153 |
| Phalloidin-iFluor™ 594 | ∼1600 | 587/603 | Texas Red | 180,000 | 0.53 | 300 Tests | $153 |
| Phalloidin-iFluor™ 633 | 1703.99 | 638/652 | Texas Red | 250,000 | 0.29 | 300 Tests | $153 |
| Phalloidin-iFluor™ 647 | 1632.87 | 654/669 | Cy5 | 250,000 | 0.25 | 300 Tests | $153 |
| Phalloidin-iFluor™ 680 | ∼2000 | 683/700 | Cy5.5 | 220,000 | 0.23 | 300 Tests | $310 |
- Extinction Coefficient = molar extinction coefficient at their maximum absorption wavelength (Units = cm-1M-1).
- Quantum Yield = fluorescence quantum yield in aqueous buffer (pH 7.2).
Phalloidin-AF Dye Conjugates
The following table outlines the fluorescence properties of available Phalloidin-AF dye conjugates for use in fluorescence imaging applications. For additional information on Phalloidin-AF dye conjugates
Table 3. Available Phalloidin-AF dye conjugates.
| Conjugate ▲ ▼ | Mol. Wt. ▲ ▼ | Ex/ Em (nm) ▲ ▼ | Filter Set ▲ ▼ | Extinction Coefficient¹ ▲ ▼ | Quantum Yield² ▲ ▼ | Unit Size ▲ ▼ | Cat No. ▲ ▼ |
| XFD350 Phalloidin *XFD350 Same Structure to Alexa Fluor™ 350* | ∼1100 | 343/441 | DAPI | 19000 | Not Determined | 300 Tests | 23150 |
| XFD488 Phalloidin *XFD488 Same Structure to Alexa Fluor™ 488* | ∼1300 | 499/520 | FITC | 73000 | 0.92 | 300 Tests | 23153 |
| XFD594 Phalloidin *XFD594 Same Structure to Alexa Fluor™ 594* | ∼1600 | 590/618 | Texas Red | 92000 | 0.66 | 300 Tests | 23158 |
- Extinction Coefficient = molar extinction coefficient at their maximum absorption wavelength (Units = cm-1M-1).
- Quantum Yield = fluorescence quantum yield in aqueous buffer (pH 7.2).
Classic Dye-Labeled Phalloidin Conjugates
The following table outlines the fluorescence properties of available Phalloidin-AF dye conjugates for use in fluorescence imaging applications. For additional information on Phalloidin-AF dye conjugates
Table 4. Available Classic Dye-Labeled Phalloidin Conjugates.
| Conjugate ▲ ▼ | Mol. Wt. ▲ ▼ | Ex/ Em (nm) ▲ ▼ | Filter Set ▲ ▼ | Extinction Coefficient¹ ▲ ▼ | Quantum Yield² ▲ ▼ | Unit Size ▲ ▼ | Cat No. ▲ ▼ |
| Phalloidin-AMCA Conjugate | ∼1000 | 346/434 | DAPI | 19000 | Not Determined | 300 Tests | 23100 |
| Phalloidin-Fluorescein Conjugate | ∼1100 | 498/517 | FITC | 80000 | 0.79 | 300 Tests | 23101 |
| Phalloidin-Tetramethylrhodamine Conjugate | ∼1300 | 552/578 | TRITC | 90000 | Not Determined | 300 Tests | 23102 |
| Phalloidin-California Red Conjugate | ∼1000 | 592 | 609 | 100000 | Not Determined | 300 Tests | 23103 |
- Extinction Coefficient = molar extinction coefficient at their maximum absorption wavelength (Units = cm-1M-1).
- Quantum Yield = fluorescence quantum yield in aqueous buffer (pH 7.2).