Phalloidin is a cyclic heptapeptide toxin isolated from the poisonous mushroom Amanita phalloides. It selectively binds filamentous actin (F-actin) with high affinity (Kd = 20 nM), yet exhibits no binding to globular monomeric actin (G-actin). Phalloidin derivatives bind actin filaments of all sizes with comparable affinity. In both muscle and non-muscle cells derived from animals and plants, phalloidin binds actin at a stoichiometry of approximately one phalloidin molecule per actin subunit. Non-specific binding is nearly negligible, resulting in clear discrimination between stained and unstained regions. Accordingly, phalloidin derivatives serve as excellent alternatives to actin antibodies for relevant research. Binding of phalloidin inhibits depolymerization of F-actin (microfilaments) and stabilizes filament structures, disrupting the dynamic balance of actin polymerization and depolymerization. This property lowers the critical concentration (Cc) required for actin polymerization to below 1 μg/mL, making phalloidin an actin polymerization enhancer. In addition, phalloidin suppresses the ATP hydrolysis activity of F-actin.
Applications:
Phalloidin and its derivatives stain F-actin at nanomolar concentrations, acting as highly practical and convenient probes for specific fluorescent labeling of F-actin in tissue sections and cultured cells. Furthermore, phalloidin and its derivatives are compact molecules with a diameter of 1.2–1.5 nm and molecular weight below 2000 Da. F-actin labeled with phalloidin retains most native biological functions of unlabeled actin. For instance, glycerol-extracted labeled muscle fibers remain contractile, and labeled actin filaments can slide on immobilized myosin matrices.
This product is LumiDye™ 555-conjugated phalloidin. It delivers highly specific staining with superior contrast and outperforms actin antibodies in labeling performance, suitable for qualitative and quantitative detection of F-actin. F-actin bound by this reagent preserves most intrinsic biological properties of actin. Its binding activity shows no species bias, supporting broad application across various biological samples.
Molecular Weight
| ~1900 | Maximum Excitation / Emission Wavelength (Ex/Em)
| Ex: 555nm; Em: 565nm | Product Appearance
| Red powder (Lyophilized solid)
| Storage Conditions
| -20℃. Protect from light. Store dry. Keep airtight.
| Solubility (25°C)
| Soluble in DMSO.
| Peptide Sequence
| LumiDye™ 555-bicyclic(Ala-DThr-Cys-cis-4-hydroxy-Pro-Ala-2-mercapto-Trp-4-hydroxy-5-amino-Leu)(S-3to6) |
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Operating Protocol (For Reference Only)
1. Preparation of Staining Solution
① Preparation of Stock Solution
Equilibrate the product to room temperature and perform brief centrifugation before use. Add 30 μL DMSO for complete dissolution and mix thoroughly to prepare 1000× LumiDye™ 555-conjugated phalloidin stock solution. Aliquot the stock solution according to experimental requirements and store at -20 °C, protected from light and moisture.
② Preparation of Working Solution
Pipette 1 μL LumiDye™ 555-conjugated phalloidin stock solution into 1 mL PBS buffer containing 1% BSA to prepare 1× working solution.
Note: Staining performance varies among different cell types; the dosage of LumiDye™ 555 phalloidin should be adjusted on a case-by-case basis.
2. Staining Procedures
① Culture cells on coverslips for more than 24 hours until cell confluency reaches 50–60%.
② Aspirate the culture medium, then wash cells twice with pre-warmed 1× PBS (pH 7.4) at 37 °C.
③ Fix cells with 4% paraformaldehyde solution prepared in PBS at room temperature for 10–30 min.
Note: Fixatives containing methanol should be avoided, as methanol may disrupt actin filaments during fixation.
④ Wash cells with PBS 2–3 times at room temperature, 10 min per wash.
⑤ Permeabilize cells at room temperature for 5 min either by dehydration with pre-chilled acetone (-20 °C) or incubation in 0.5% Triton X-100 solution.
⑥ Wash cells with PBS 2–3 times at room temperature, 10 min per wash.
⑦ Add 100 μL prepared LumiDye™ 555-conjugated phalloidin working solution per well (96-well plate) to fully cover cells on the coverslip. Incubate at room temperature away from light for 30 min (incubation at 4 °C to 37 °C is acceptable for general applications).
Note: To reduce background fluorescence, supplement the LumiDye™ 555-conjugated phalloidin working solution with 1% BSA. Place coverslips in a sealed container during incubation to prevent liquid evaporation.
⑧ Rinse coverslips with PBS 3 times, 5 min per rinse.
⑨ Counterstain cell nuclei with 100 μL ready-to-use DAPI solution (100 nM) per well (96-well plate) for approximately 30 seconds.
⑩ Rinse coverslips with PBS, then invert them onto glass slides pre-dropped with mounting medium. Gently blot excess mounting medium with filter paper and permanently seal slides with clear nail polish. Prepared slides can be stored at 4 °C protected from light, and F-actin fluorescence analysis can normally be conducted within 6 months.
⑪ Observe fluorescence signals under a fluorescence microscope or confocal laser scanning microscope. Select the LumiDye™ 555 filter set (Ex/Em = 555/565 nm) and DAPI filter set (Ex/Em = 364/454 nm) for imaging.
Materials to Be Prepared by User
1. Reagents: Methanol, 1× PBS buffer (pH 7.4, cell culture grade), 4% paraformaldehyde fixative (dissolved in PBS), acetone or 0.5% Triton X-100 permeabilization solution (dissolved in PBS), DAPI, mounting medium, standard-grade BSA, sealing agent for coverslips (e.g., clear nail polish).
2. Instruments & Consumables: Glass slides and coverslips; fluorescence microscope or confocal laser scanning microscope equipped with LumiDye™ 555 excitation/emission filter set and DAPI excitation/emission filter set.
Precautions:
1. Definition of one unit (T) of fluorescently labeled phalloidin: At the recommended working concentration of 200 nM with 100 μL staining solution per assay, 300 staining tests can be performed. At a working concentration of 100 nM with 200 μL staining solution per assay, 300 staining tests can also be completed.
2. Phalloidin is toxic. Handle with great care.
3. This product is supplied as lyophilized powder with a very small mass which is barely visible. Briefly centrifuge the vial before use, then dissolve with solvent. The resulting solution is nearly colorless.
4. Wear a lab coat and disposable gloves during operation for personal safety and health protection.
5. Our company only provides partial technical information for some products. We do not guarantee the authenticity and authority of the above data. All data are for research reference only.
