鬼笔环肽（Phalloidin）是一种来源于毒蕈类鬼笔鹅膏（Amanitaphalloides）的环状七肽毒素，以高亲和力（Kd=20nM）选择性结合于丝状肌动蛋白F-actin，而不会与单体肌动蛋白G-actin结合。鬼笔环肽衍生物也以相近的亲和力结合于大小纤维，无论是动植物来源的肌肉细胞或非肌肉细胞，按照每一个肌动蛋白亚基约与一个鬼笔环肽分子的计量比结合。且非特异性结合几乎可忽略，染色区域和非染色区域辨识度非常明显。因此，鬼笔环肽衍生物特别适合替代肌动蛋白（Actin）抗体进行相关研究。鬼笔环肽的结合阻止丝状肌动蛋白（微丝）的解离，稳定微丝结构，从而破坏微丝的聚合-去聚合的动态平衡。此特性使得肌动蛋白聚合发生的临界浓度（CC）降至<1μg/mL，因此，可用作一种聚合促进剂。此外，鬼笔环肽还可抑制F-actin的ATP水解活性。
鬼笔环肽及其衍生物在纳摩尔浓度即可对F-actin染色，是非常实用和方便的探针，通常用于组织切片或细胞培养物中 F-actin的特异性荧光染色。另外，鬼笔环肽及其衍生物很小，直径约1.2~1.5nm，分子量<2000Da，经标记后的F-actin仍维持许多标记前的功能。比如，标记的甘油抽提肌纤维仍能收缩；标记的肌动蛋白丝仍能在固相肌球蛋白基质中移动。
本品为TRITC (四甲基异硫氰酸罗丹明) 标记的鬼笔环肽，染色反应特异性强，对比性高，具有比Actin抗体更好的染色效果，适合用作F-actin的定性和定量检测。另外，经本品结合后的F-actin仍能维持actin自身具有的许多生物学特性，且本品的结合没有物种差异性，适用性广泛。
| CAS No. | 915013-10-4 | | 分子量 | 1231.4
| | 最大激发/发射波长 (Ex/Em) | Ex: 540-546nm; Em: 565-575nm
| | 产品外观 | 红色粉末 (冻干粉)
| | 储存条件 | -20℃，避光防潮密闭干燥 | | 溶解性 (25℃) | 溶于DMSO、DMF、甲醇或者乙腈水溶液 (20%)
| | 多肽序列 | TRITC-bicyclic(Ala-DThr-Cys-cis-4-hydroxy-Pro-Ala-2-mercapto-Trp-4-hydroxy-5-amino-Leu)(S-3to6)
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使用方法：（仅供参考）
1. 染色液的配制
① 母液的配制：使用前将本品恢复至室温并简短离心，加入30μL DMSO使其充分溶解，混匀即可获得1000×罗丹明标记鬼笔环肽母液。根据实验情况，对其分装并于-20℃避光干燥保存。
② 工作液的配制：吸取1µL 罗丹明标记鬼笔环肽母液至1mL PBS（含1%BSA）缓冲液中即可得到1×工作液。
注意：不同的细胞染色情况不同，相应TRITC鬼笔环肽使用量也需根据不同情况而定。推荐工作浓度为：80~200nM。工作液现配现用。
2. 染色步骤
① 细胞爬片生长>24h，使其密度达到50~60%汇合度。
② 吸掉培养液，37℃预热的1×PBS（pH7.4）清洗细胞2次。
③ 使用溶于PBS的4%甲醛溶液进行细胞固定，室温固定10~30min。
注意：避免固定剂中含有甲醇成分，因为甲醇在固定过程中可能破坏肌动蛋白。
④ 室温条件下，用PBS清洗细胞2~3次，每次10min。
⑤ 室温条件下，用丙酮（-20℃预冷）脱水或者用0.5% Triton X-100溶液透化处理5min。
⑥ 室温条件下，用PBS清洗细胞2~3次，每次10min。
⑦ 取100µL/孔（96孔板）配制好的罗丹明标记鬼笔环肽工作液，覆盖住盖玻片上的细胞，室温避光孵育30min（通常情况下，4℃~37℃孵育皆可）。
注意：为了降低背景，可于罗丹明标记的鬼笔环肽工作液内加入1%BSA；另外，孵育过程中为了避免溶液挥发，可将盖玻片转移到一个密封的容器内。
⑧ 用PBS清洗盖玻片3次，每次5min。
⑨ 使用100µL/孔（96孔板）即用型DAPI溶液（浓度：100nM）对细胞核进行复染，约30s。
⑩ 用PBS清洗盖玻片，然后倒置在已经滴有封片剂的载玻片上。使用纸巾轻轻擦掉多余封片剂，然后用指甲油永久封片。此法制备的标本玻片可置于4℃避光保存，通常6个月内可继续做F-actin染色分析。
⑪ 荧光显微镜或者共聚焦显微镜下进行荧光观察，选择TRITC激发/发射滤片 (Ex/Em=545/570nm) 和DAPI 激发/发射滤片 (Ex/Em=364/454nm)。
需要自备材料：
1. 试剂：甲醇、1×PBS缓冲液，pH7.4，细胞培养级别、固定液4%多聚甲醛（溶于PBS缓冲液）、丙酮或透化液0.5%Triton X-100（溶于PBS缓冲液）、DAPI、封片剂、BSA（标准级别）、盖玻片周围密封液（如透明指甲油）。
2. 仪器及耗材：载玻片和盖玻片、组装有TRITC激发/发射滤片，以及DAPI激发/发射滤片的荧光显微镜或共聚焦显微镜。
注意事项：
1. 荧光标记鬼笔环肽的一个单位(T)的定义：按照推荐工作液浓度200nM，每次用量为100μL染色工作液时，可检测的次数300次；按照工作液浓度100nM，每次用量为200μL染色工作液时，可检测的次数也是300次。
2. 鬼笔环肽具有毒性，需小心操作。
3. 本产品为冻干粉形式，微量不易观察。使用前瞬时离心，加溶剂溶解后使用，溶解后为淡粉色透明液体。
4. 为了您的安全和健康，请穿实验服并戴一次性手套操作。
5. 部分产品我司仅能提供部分信息，我司不保证所提供信息的权威性，以上数据仅供参考交流研究之用。
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 TRITC (tetramethylrhodamine isothiocyanate)-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.
| CAS No. | 915013-10-4 | Molecular Weight
| 1231.4
| Maximum Excitation / Emission Wavelength (Ex/Em)
| Ex:540-546nm; Em:565-575nm
| Product Appearance
| Red powder (Lyophilized solid)
| Storage Conditions
| -20℃. Protect from light. Store dry. Keep airtight.
| Solubility (25°C)
| Soluble in DMSO, DMF, methanol or 20% aqueous acetonitrile solution
| Peptide Sequence
| TRITC-bicyclic(Ala-DThr-Cys-cis-4-hydroxy-Pro-Ala-2-mercapto-Trp-4-hydroxy-5-amino-Leu)(S-3to6)
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Protocol (For Reference Only)
1. Preparation of Staining Solution
① Stock Solution Preparation
Equilibrate the product to room temperature and perform brief centrifugation before use. Add 30 μL DMSO to fully dissolve the powder and mix well to prepare 1000× TRITC-conjugated phalloidin stock solution. Aliquot according to experimental requirements and store at -20 °C, protected from light and moisture.
② Working Solution Preparation
Pipette 1 μL TRITC-conjugated phalloidin stock solution into 1 mL PBS buffer containing 1% BSA to obtain 1× working solution.
Note: Staining performance varies among different cell types; the dosage of TRITC phalloidin should be adjusted accordingly. The recommended working concentration ranges from 80 to 200 nM. Prepare the working solution freshly right before use.
2. Staining Procedures
① Culture cells on coverslips for more than 24 hours until the cell confluency reaches 50–60%.
② Aspirate culture medium, then wash cells twice with pre-warmed 1× PBS (pH 7.4) at 37 °C.
③ Fix cells with 4% paraformaldehyde 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 by dehydration with pre-cooled acetone (-20 °C) or incubation in 0.5% Triton X-100 solution for 5 min at room temperature.
⑥ Wash cells with PBS 2–3 times at room temperature, 10 min per wash.
⑦ Add 100 μL prepared TRITC-conjugated phalloidin working solution per well (96-well plate) to fully cover cells on the coverslip. Incubate at room temperature in the dark for 30 min (incubation at 4 °C to 37 °C is also acceptable for general applications).
Note: To reduce background signal, supplement the TRITC-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 Wash coverslips with PBS, then invert them onto glass slides pre-dropped with mounting medium. Gently blot excess mounting medium with filter paper and seal slides permanently with nail polish. Prepared slides can be stored at 4 °C away from light, and F-actin fluorescence analysis can be performed within 6 months normally.
⑪ Observe fluorescence under a fluorescence microscope or confocal laser scanning microscope. Select TRITC filter set (Ex/Em = 545/570 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 TRITC 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 in a trace‑sized amount that is hardly visible. Briefly centrifuge the vial before use, dissolve it with solvent, and the resulting solution is a light‑pink transparent liquid.
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.
ZJ26F0939361
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