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| Product Name | FITC Phalloidin |
| Catalog No. | RESK-0333 |
| Description | Phalloidin is a cyclic heptapeptide toxin derived from the death cap mushroom Amanita phalloides. It binds filamentous actin (F-actin) with high affinity (Kd = 20 nM) and high selectivity, without binding monomeric actin (G-actin), and is therefore widely used to label F-actin in cell cultures or cell-free systems for qualitative and quantitative analysis. Phalloidin derivatives bind large and small filaments from both muscle and non-muscle cells of animal and plant origin at a stoichiometry of approximately one phalloidin molecule per actin subunit. Non-specific binding is negligible, giving excellent contrast between stained and unstained regions, making phalloidin conjugates an ideal alternative to actin antibodies. Because phalloidin derivatives are very small (about 12-15 angstroms in diameter, molecular weight < 2000 Da), many native properties of the labeled actin are preserved: interaction with actin-binding proteins such as myosin, tropomyosin and DNase I is maintained, labeled filaments can still penetrate solid-phase myosin matrices, and glycerol-extracted myofibrils still contract after labeling. Binding of phalloidin prevents depolymerization of microfilaments and stabilizes them, lowering the critical concentration for actin polymerization to < 1 ug/mL, so it can also serve as a polymerization promoter; it additionally inhibits the ATP hydrolysis activity of F-actin. This product is FITC-conjugated phalloidin with strong staining specificity and high contrast, providing better staining performance than actin antibodies for qualitative and quantitative F-actin detection. The binding shows no species difference and is broadly applicable. It is supplied as a 20 uM stock solution; a working concentration of 80-200 nM is recommended. |
| Intended Use | Qualitative and quantitative fluorescence labeling and analysis of filamentous actin (F-actin) in fixed cells, cell cultures and cell-free systems for cytoskeleton research. For research use only. |
| Principle / Technology | FITC-conjugated phalloidin selectively binds F-actin with high affinity (Kd = 20 nM) at a stoichiometry of about one phalloidin molecule per actin subunit, without binding G-actin, producing bright green fluorescence at actin filaments. |
| Detection Method | Fluorescence microscopy (green channel); maximum excitation/emission (Ex/Em) 495-496/513-516 nm. |
| Sample Type | Fixed cultured cells and cell-free systems. Not recommended for paraffin-embedded or frozen tissue sections (an anti-actin antibody is recommended for sections). For plant cells, the cell wall should be removed to prepare protoplasts before staining. |
| Performance Range / Specifications | 300 tests (300 T). The 300 T specification refers to approximately 300 staining assays, supplied as 300 uL of a 20 uM stock solution in methanol. Molecular formula: C56H60N10O15S2 Molecular weight: 1177.3 Ex/Em: 495-496/513-516 nm Peptide sequence: bicyclic(Ala-DThr-Cys-cis-4-hydroxy-Pro-Ala-2-mercapto-Trp-4-hydroxy-5-amino-Leu)(S-3 to 6), FITC-labeled Appearance: light yellow to yellow solution |
| Sensitivity / LOD | Binding affinity Kd = 20 nM; recommended working concentration 80-200 nM. |
| Specificity | Selectively binds F-actin and does not bind G-actin; non-specific binding is negligible. No species specificity for staining of fixed cells. Overexpression of G-actin in cells does not affect staining. |
| Reaction Conditions / Protocol | 1. Fix cells with 4% paraformaldehyde in PBS (avoid methanol-containing fixatives, as methanol may disrupt F-actin during fixation). 2. Permeabilize with 0.5% Triton X-100 for 10-15 min (for hard-to-stain cells such as macrophages, extend permeabilization or use acetone at -20 °C or below for dehydration). 3. Block with BSA to reduce background. 4. Dilute the stock to a working concentration of 80-200 nM and add enough working solution to completely cover the cells. 5. Observe by fluorescence microscopy, preferably within 60 min after staining. |
| Components / Formulation | FITC-conjugated phalloidin, supplied as a 20 uM stock solution in methanol (total volume 300 uL). |
| Storage Conditions | Store at -20 °C, protected from light and kept dry. |
| Shelf Life | 1 year when stored at -20 °C protected from light. |
| Package Specifications | 300 T |
| Product Form | Light yellow to yellow liquid (stock solution in methanol). |
| Concentration | 20 uM stock solution; recommended working concentration 80-200 nM. |
| Molecular Weight | 1177.3 Da |
| Source / Origin | Phalloidin derived from the mushroom Amanita phalloides, conjugated with FITC. |
| Shipping Conditions | Shipped with ice packs. |
| Expiration Date / Stability | Stable for 1 year at -20 °C protected from light. The fluorescence signal gradually weakens after mounting; stained samples should be observed promptly and overnight storage is not recommended. |
| Compatibility | Compatible with dual labeling, e.g., nuclear counterstaining with DAPI and co-staining with antibodies (perform antibody staining first, then phalloidin staining). Not compatible with click chemistry reactions (e.g., EdU); co-staining abolishes the phalloidin fluorescence signal. Not cell-permeable; not recommended for live-cell labeling or flow cytometry. |
| Recommended Buffer System | 4% paraformaldehyde in PBS for fixation; 0.5% Triton X-100 for permeabilization; BSA for blocking. |
| Application Notes / Precautions | If fluorescence quenches quickly, reduce the reagent concentration; use a low-energy laser to locate the field and switch to high-energy excitation for observation. Do not co-stain with click chemistry reactions (e.g., EdU). Complete observation and detection within 60 min after staining; mounted slides are not recommended for later re-analysis. For macrophages and other poorly permeabilized cells, extend permeabilization time. Use methanol-free fixatives, as methanol may destroy F-actin. For plant cells, remove the cell wall to prepare protoplasts before staining. |
| Recognition Site | Filamentous actin (F-actin); approximately one phalloidin molecule binds per actin subunit. |
For research use only, not for clinical use.
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