Phalloidin Conjugates

This green fluorescent phalloidin conjugate (equivalent to FITC labeled phalloidin) selectively binds to F-actins. Used at nanomolar concentrations, phalloidin derivatives are convenient probes for labeling, identifying and quantitating F-actins in formaldehyde-fixed and permeabilized tissue sections, cell cultures or cell-free experiments. Phalloidin 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, essentially stabilizing actin filaments through the prevention of filament depolymerization. Moreover, phalloidin is found to inhibit the ATP hydrolysis activity of F-actin. Phalloidin functions differently at various concentrations in cells. When introduced into the cytoplasm at low concentrations, phalloidin recruits the less polymerized forms of cytoplasmic actin as well as filamin into stable "islands" of aggregated actin polymers, yet it does not interfere with stress fibers, i.e. thick bundles of microfilaments. The property of phalloidin is a useful tool for investigating the distribution of F-actin in cells by labeling phalloidin with fluorescent analogs and using them to stain actin filaments for light microscopy. Fluorescent derivatives of phalloidin have turned out to be enormously useful in localizing actin filaments in living or fixed cells as well as for visualizing individual actin filaments in vitro. Fluorescent phalloidin derivatives have been used as an important tool in the study of actin networks at high resolution. AAT Bioquest offers a variety of fluorescent phalloidin derivatives with different colors for multicolor imaging applications.

Example protocol

AT A GLANCE

Protocol Summary

Prepare samples in microplate wells
Remove liquid from samples in the plate
Add Phalloidin-Fluorescein Conjugate solution (100 μL/well)
Stain the cells at room temperature for 20 to 90 minutes
Wash the cells
Examine the specimen under microscope with FITC filter

Important Warm the vial to room temperature and centrifuge briefly before opening.

Storage and Handling Conditions

The solution should be stable for at least 6 months if store at -20 °C. Protect the fluorescent conjugates from light, and avoid freeze/thaw cycles.
Note Phalloidin is toxic, although the amount of toxin present in a vial could be lethal only to a mosquito (LD50 of phalloidin = 2 mg/kg), it should be handled with care.

PREPARATION OF WORKING SOLUTION

Phalloidin-Fluorescein Conjugate working solution

Add 1 µL of Phalloidin-Fluorescein Conjugate solution to 1 mL of PBS with 1% BSA.
Note The stock solution of phalloidin conjugate should be aliquoted and stored at -20 °C. protected from light.
Note Different cell types might be stained differently. The concentration of phalloidin conjugate working solution should be prepared accordingly.

SAMPLE EXPERIMENTAL PROTOCOL

Stain the cells

Perform formaldehyde fixation. Incubate cells with 3.0–4.0 % formaldehyde in PBS at room temperature for 10–30 minutes.
Note Avoid any methanol containing fixatives since methanol can disrupt actin during the fixation process. The preferred fixative is methanol-free formaldehyde.
Rinse the fixed cells 2–3 times in PBS.
Optional: Add 0.1% Triton X-100 in PBS into fixed cells for 3 to 5 minutes to increase permeability. Rinse the cells 2–3 times in PBS.
Add 100 μL/well (96-well plate) of Phalloidin-Fluorescein Conjugate working solution into the fixed cells, and stain the cells at room temperature for 20 to 90 minutes.
Rinse cells gently with PBS 2 to 3 times to remove excess phalloidin conjugate before plating, sealing and imaging under microscope with FITC filter set.

Spectrum

Open in Advanced Spectrum Viewer

Product family

Name	Excitation (nm)	Emission (nm)	Extinction coefficient (cm ^-1 M ^-1)	Correction Factor (280 nm)
Phalloidin-AMCA Conjugate	346	434	19000	0.153
Phalloidin-Tetramethylrhodamine Conjugate	552	578	90000	0.178

Citations

View all 49 citations: Citation Explorer

Hypoxia-and Postirradiation reoxygenation-induced HMHA1/ARHGAP45 expression contributes to cancer cell invasion in a HIF-dependent manner
Authors: Lee, Peter WT and Suwa, Tatsuya and Kobayashi, Minoru and Yang, Hui and Koseki, Lina R and Takeuchi, Satoshi and Chow, Christalle CT and Yasuhara, Takaaki and Harada, Hiroshi
Journal: British Journal of Cancer (2024): 1--12

MiR-145 regulates steroidogenesis in mouse primary granulosa cells by targeting Arpc5 and subsequent cytoskeleton remodeling
Authors: MA, Lanfang and WANG, Shuo and YANG, Jun and TANG, Weicheng and WU, Zhangying and CAO, Lili and LUO, Aiyue and FU, Fangfang and YANG, Shuhong and WANG, Shixuan
Journal: Journal of Reproduction and Development (2023): 2022--137

Sirt1 Regulates Corneal Epithelial Migration by Deacetylating Cortactin
Authors: Lin, Yong and Liu, Qi and Li, Li and Yang, Rusen and Ye, Juxiu and Yang, Shuai and Luo, Guangying and Reinach, Peter S and Yan, Dongsheng
Journal: Investigative Ophthalmology \& Visual Science (2022): 14--14

2-Mercaptoethanol promotes porcine oocyte maturation in vitro by maintaining autophagy homeostasis
Authors: Zhang, Yaping and Li, Qiqi and Li, Wangchang and Yan, Ke and Liu, Yaru and Xu, Huiyan and Jiang, Mingsheng and Lu, Yangqing and Liang, Xingwei and Shang, Jianghua and others,
Journal: Theriogenology (2022): 155--167

Allicin protects porcine oocytes against LPS-induced defects during maturation in vitro
Authors: Li, Qiqi and Zhang, Yaping and Li, Wangchang and Yan, Ke and Liu, Yaru and Xu, Huiyan and Lu, Yangqing and Liang, Xingwei and Yang, Xiaogan
Journal: Theriogenology (2022): 138--147

References

View all 127 references: Citation Explorer

Improved penile histology by phalloidin stain: circular and longitudinal cavernous smooth muscles, dual-endothelium arteries, and erectile dysfunction-associated changes
Authors: Lin G, Qiu X, F and el TM, Albersen M, Wang Z, Lue TF, Lin CS.
Journal: Urology (2011): 970 e1

Phalloidin perturbs the interaction of human non-muscle myosin isoforms 2A and 2C1 with F-actin
Authors: Diensthuber RP, Muller M, Heissler SM, Taft MH, Chizhov I, Manstein DJ.
Journal: FEBS Lett (2011): 767

pH-(low)-insertion-peptide (pHLIP) translocation of membrane impermeable phalloidin toxin inhibits cancer cell proliferation
Authors: An M, Wijesinghe D, Andreev OA, Reshetnyak YK, Engelman DM.
Journal: Proc Natl Acad Sci U S A (2010): 20246

Labeling cytoskeletal F-actin with rhodamine phalloidin or fluorescein phalloidin for imaging
Authors: Chazotte B., undefined
Journal: Cold Spring Harb Protoc (2010): pdb prot4947

Effect of Phalloidin on Filaments Polymerized from Heart Muscle Adp-Actin Monomers
Authors: Vig A, Dudas R, Kupi T, Orban J, Hild G, Lorinczy D, Nyitrai M.
Journal: J Therm Anal Calorim (2009): 721

Page updated on October 9, 2024

Ordering information

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Catalog Number	231002310123102231032311023111231152311623117231192312223125231272312823129231302313123140231502315323158231595301
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Physical properties

Molecular weight	~1100
Solvent	DMSO

Spectral properties

Absorbance (nm)	487
Correction Factor (260 nm)	0.32
Correction Factor (280 nm)	0.35
Extinction coefficient (cm ^-1 M ^-1)	80000¹
Excitation (nm)	498
Emission (nm)	517
Quantum yield	0.7900¹, 0.95²

Storage, safety and handling

H-phrase	H301, H311, H331
Hazard symbol	T
Intended use	Research Use Only (RUO)
R-phrase	R23, R24, R25
Storage	Freeze (< -15 °C); Minimize light exposure
UNSPSC	12352200