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| 25mg | ||
| 50mg | ||
| Other Sizes |
Phalloidin [Mast Cell Degranulating (MCD) Peptide] is a novel and potent toxin derived from mushroom and may be used to label F-actin of the cytoskeleton with fluorochrome (λex=495 nm, λem=520 nm). It has a sequence of Ala-{d-Thr}-Cys-{Hyp}-Ala-Trp-Leu (Disulfide bridge: Cys3-Cys6); and sequence shortening of A-{d-Thr}-C-{Hyp}-AWL (Disulfide bridge: Cys3-Cys6).
| Targets |
Fluorescent Dye; F-actin
|
|---|---|
| ln Vitro |
Phalloidin staining works well on cells fixed in PBS containing 0.2% glutaraldehyde, but not on fixed cells (possibly because of defects in disrupted actin filaments) [1].
The eukaryotic cell has evolved to compartmentalize its functions and transport various metabolites among cellular compartments. Therefore, in cell biology, the study of organization and structure/function relationships is of great importance. The cytoskeleton is composed of a series of filamentous structures, including intermediate filaments, actin filaments, and microtubules. Immunofluorescent staining has been most frequently used to study cytoskeletal components. However, it is also possible to fluorescently label isolated cytoskeletal proteins and either microinject them back into the cell or add them to fixed, permeabilized cells. Alternatively, it is possible to use the mushroom-derived fluorescinated toxins, phalloidin or phallacidin, to label F-actin of the cytoskeleton, as is described in this article. Phalloidin is available labeled with different fluorophores. The choice of the specific fluorophore should depend on whether phalloidin labeling for actin is part of a double-label experiment. In most cells, the abundance of actin filaments should provide a very strong signal. In double-label experiments, the fluorophore should be chosen to take this into account. In general, rhodamine labels are more resistant to photobleaching and can be subjected to the longer exposures required for finer structures[1]. |
| Cell Assay |
Phalloidin Staining Protocol [1]
1. Dilution: Prepare a 1:200 dilution of fluorescein or rhodamine Phalloidin (300 units/mL stock) in PBS. 2. Washing: Aspirate the cell culture medium from cells grown on glass coverslips and rinse three times with PBS. 3. Fixation: Fix cells for 10 minutes in 3.7% formaldehyde solution. 4. Post-Fixation Wash: Rinse fixed cells three times with PBS (5 min per wash). 5. Permeabilization: Treat cells with 0.2% Triton X-100 for 5 minutes. 6. Post-Permeabilization Wash: Rinse cells three times with PBS. 7. Staining: Incubate cells with fluorescein or rhodamine Phalloidin for 5–10 minutes at room temperature. 8. Final Wash: Rinse cells three times with PBS (5 min per wash). 9. Mounting & Imaging: Mount coverslips and observe cells using the appropriate filter set (fluorescein or rhodamine, depending on the probe). |
| ADME/Pharmacokinetics |
Absorption, Distribution and Excretion
THE UPTAKE, BINDING AND ELIMINATION OF PHALLOIDIN BY LIVER WAS COMPARED IN ADULT AND BABY (17-19 DAYS OLD) RATS IN VIVO AND IN VITRO. IN BOTH GROUPS, THERE WAS NO RELATION BETWEEN THE CONCENTRATION OF THE POISON IN THE LIVER AND THE TOXICITY. ALTHOUGH BABY RATS SHOWED A SIGNIFICANTLY HIGHER TOLERANCE AGAINST PHALLOIDIN THAN THE ADULT ANIMALS, THE CONCENTRATION OF THE POISON IN THE LIVER OF BABY RATS WAS HIGHER, AND THE ELIMINATION WAS SIGNIFICANTLY SLOWER THAN IN ADULT RATS. THE VERY TIGHT BINDING AND CONCENTRATION OF PHALLOIDIN IN THE LIVER WAS EXPLAINED BY AN EXTREMELY LOW DISSOCIATION CONSTANT. PHALLOIN AND GAMMA-AMANITIN WERE DETECTED WITHIN THE FIRST DAY AND PHALLOIDIN WITHIN THE SECOND DAY IN THE URINE OF RATS AFTER ADMINISTRATION OF AMANITA PHALLOIDES EXTRACTS. Metabolism / Metabolites VARIOUS OBSERVATIONS SUGGESTED THAT PHALLOIDINE, A POISONOUS CONSTITUENT OF THE GREEN MUSHROOM, AMANITA PHALLOIDES, WOULD NOT BE TOXIC PER SE, BUT WOULD UNDERGO BIOACTIVATION IN THE LIVER. USING (3)H-DEMETHYLPHALLOIN, A TOXIC DERIVATIVE OF PHALLOIDINE, /IT WAS CONCLUDED/...THAT (3)H-DEMETHYLPHALLOIN, AND MOST PROBABLY ALSO PHALLOIDINE, ARE UNMETABOLIZED IN THE LIVERS OF RATS AND MICE. A SECONDARY EXCRETORY PRODUCT IN THE URINE OF TREATED ANIMALS WAS FOUND TO BE AN AUTODEGRADATION COMPOUND OF THE (3)H-LABELED DERIVATIVE. |
| Toxicity/Toxicokinetics |
Interactions
PRETREATMENT WITH RIFAMPICIN (100 OR 300 MG/KG, ORAL) PROTECTED MICE AGAINST THE TOXICITY OF PHALLOIDIN (3 MG/KG, IP) AS REFLECTED BY THE DECREASE IN THE MORTALITY RATE. IN MICE, THE ACUTE HEMORRHAGIC NECROSIS OF THE LIVER INDUCED BY PHALLOIDIN WAS PREVENTED BY TREATMENT WITH SILYBIN, A PLANT COMPOUND ISOLATED FROM SILYBUM MARIANUM. PRETREATMENT WITH A SINGLE DOSE OF SILYBIN COMPLETELY ABOLISHED THE MORPHOLOGIC CHANGES INDUCED BY THE TOXIN, AND SIGNIFICANTLY DECREASED THE ACTIVITIES OF SERUM ENZYMES. Toxicity Summary It binds actin, preventing its depolymerization and poisoning the cell. Phalloidin binds specifically at the interface between F-actin subunits, locking adjacent subunits together. Phalloidin, a bicyclic heptapeptide, binds to actin filaments much more tightly than to actin monomers, leading to a decrease in the rate constant for the dissociation of actin subunits from filament ends, which essentially stabilizes actin filaments through the prevention of filament depolymerization. Moreover, phalloidin is found to inhibit the ATP hydrolysis activity of F-actin (L1183). LD50: 2 mg/kg (Mouse) (T258) |
| References | |
| Additional Infomation |
Phalloidin is a homodetic bicyclic heptapeptide having a sulfide bridge.
Phalloidine has been reported in Amanita suballiacea, Amanita phalloides, and other organisms with data available. Very toxic polypeptide isolated mainly from AMANITA phalloides (Agaricaceae) or death cup; causes fatal liver, kidney and CNS damage in mushroom poisoning; used in the study of liver damage. Mechanism of Action IN CONTRAST TO NORMAL LIVER CELLS, AS-30D RAT HEPATOMA CELLS ARE INSENSITIVE TO PHALLOIDIN. HEPATOMA CELLS APPARENTLY DO NOT CONSUME THE TOXIN AS DO NORMAL LIVER CELLS. TOLERATED DOSES OF PHALLOIDIN PROTECT MICE AGAINST LETHAL DOSES OF PHALLOIDIN. RESISTANCE IS CONFERRED BY THE 1/10 LD95 OF PHALLOIDIN AND SETS IN AT ABOUT 8 HOURS AFTER PRETREATMENT. WITHIN 1 HOUR OF EXPOSURE OF PRIMARY CULTURES OF ADULT RAT HEPATOCYTES TO PHALLOIDIN AT 50 MUG/ML, 60-70% OF THE CELLS WERE DEAD (TRYPAN BLUE-STAINABLE). THERE WAS NO LOSS OF VIABILITY OF THE SAME CELLS EXPOSED TO PHALLOIDIN IN CULTURE MEDIUM DEVOID OF CA2+. INITIALLY PHALLOIDIN INTERACTS IN A CA2+-INDEPENDENT PROCESS WITH CELL MEMBRANE-ASSOCIATED ACTIN. THE 2ND STEP IS A CA2+-DEPENDENT PROCESS THAT MOST LIKELY REPRESENTS AN INCREASED INFLUX OF CA2+ ACROSS A COMPROMISED CELL MEMBRANE PERMEABILITY BARRIER AND DOWN THE STEEP CONCENTRATION GRADIENT THAT EXISTS BETWEEN THE OUTSIDE AND INSIDE OF THE CELL. THESE RESULTS STRENGTHEN THE HYPOTHESIS THAT DISTURBANCES IN CA2+ HOMEOSTASIS ARE INDUCED IN VIVO. PHALLOIDIN ADMINISTERED TO MALE RATS FOR 7 DAYS (500 MUG/KG/DAY) INCREASED THE MEAN HEPATIC CONTENT OF FILAMENTOUS ACTIN. BOTH BILE FLOW AND BILE ACID EXCRETION DIMINISHED PROPORTIONALLY. MICROFILAMENTS MAY INFLUENCE THE PERMEABILITY OF TIGHT JUNCTIONS BETWEEN HEPATOCYTES. BILE CONSTITUENTS MIGHT REFLUX FROM THE CANALICULUS TO THE INTERCELLULAR SPACE IN PHALLOIDIN-INDUCED CHOLESTASIS. |
| Molecular Formula |
C35H48N8O12S
|
|---|---|
| Molecular Weight |
804.867020000001
|
| Exact Mass |
788.316
|
| CAS # |
17466-45-4
|
| PubChem CID |
441542
|
| Appearance |
White to off-white solid powder
|
| Density |
1.51g/cm3
|
| Boiling Point |
1370.5ºC at 760mmHg
|
| Melting Point |
MAXIMUM ABSORPTION (WATER): 295 NM (E= 0.597, 1%, 1 CM); MELTING POINT: 280-282 °C /HEXAHYDRATE/
|
| Flash Point |
782.6ºC
|
| Vapour Pressure |
0mmHg at 25°C
|
| Index of Refraction |
1.683
|
| LogP |
-1.7
|
| Hydrogen Bond Donor Count |
11
|
| Hydrogen Bond Acceptor Count |
12
|
| Rotatable Bond Count |
4
|
| Heavy Atom Count |
55
|
| Complexity |
1510
|
| Defined Atom Stereocenter Count |
10
|
| SMILES |
C[C@H]1C(=O)N[C@H]2CC3=C(NC4=CC=CC=C34)SC[C@@H](C(=O)N5C[C@H](C[C@H]5C(=O)N1)O)NC(=O)[C@H](NC(=O)[C@@H](NC(=O)[C@@H](NC2=O)C[C@](C)(CO)O)C)[C@H](C)O
|
| InChi Key |
KPKZJLCSROULON-QKGLWVMZSA-N
|
| InChi Code |
InChI=1S/C35H48N8O11S/c1-15-27(47)38-22-10-20-19-7-5-6-8-21(19)41-33(20)55-13-24(34(53)43-12-18(46)9-25(43)31(51)37-15)40-32(52)26(17(3)45)42-28(48)16(2)36-30(50)23(39-29(22)49)11-35(4,54)14-44/h5-8,15-18,22-26,41,44-46,54H,9-14H2,1-4H3,(H,36,50)(H,37,51)(H,38,47)(H,39,49)(H,40,52)(H,42,48)/t15-,16-,17-,18-,22-,23-,24-,25-,26+,35+/m0/s1
|
| Chemical Name |
(1S,14R,18S,20S,23S,28S,31S,34R)-28-[(2R)-2,3-dihydroxy-2-methylpropyl]-18-hydroxy-34-[(1S)-1-hydroxyethyl]-23,31-dimethyl-12-thia-10,16,22,25,27,30,33,36-octazapentacyclo[12.11.11.03,11.04,9.016,20]hexatriaconta-3(11),4,6,8-tetraene-15,21,24,26,29,32,35-heptone
|
| Synonyms |
17466-45-4; Phalloidin from Amanita phalloides; 28-(2,3-Dihydroxy-2-methylpropyl)-18-hydroxy-34-(1-hydroxyethyl)-23,31-dimethyl-12-thia-10,16,22,25,27,30,33,36-octazapentacyclo[12.11.11.03,11.04,9.016,20]hexatriaconta-3(11),4,6,8-tetraene-15,21,24,26,29,32,35-heptone; SCHEMBL39329; GTPL4736; KPKZJLCSROULON-UHFFFAOYSA-N; NSC523214; SMP1_000234;
|
| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light. |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
|
| Solubility (In Vitro) |
May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples
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|---|---|
| Solubility (In Vivo) |
Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.
Injection Formulations
Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution. Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline) Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil) Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals). View More
Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)] Oral Formulations
Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium) Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals). View More
Oral Formulation 3: Dissolved in PEG400 (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.2424 mL | 6.2122 mL | 12.4244 mL | |
| 5 mM | 0.2485 mL | 1.2424 mL | 2.4849 mL | |
| 10 mM | 0.1242 mL | 0.6212 mL | 1.2424 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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