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AAT Bioquest

Phalloidin Conjugates

This NIR fluorescent phalloidin conjugate selectively binds to F-actins with much higher photostability than the fluorescein-phalloidin conjugates. 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
  1. Prepare samples in microplate wells
  2. Remove liquid from samples in the plate
  3. Add Phalloidin-iFluor™ 680 Conjugate solution (100 μL/well)
  4. Stain the cells at room temperature for 20 to 90 minutes
  5. Wash the cells
  6. Examine the specimen under microscope with Cy5.5 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 STOCK SOLUTIONS

Unless otherwise noted, all unused stock solutions should be divided into single-use aliquots and stored at -20 °C after preparation. Avoid repeated freeze-thaw cycles.

Phalloidin-iFluor™ 680 Conjugate stock solution
Add 30 µL of DMSO into the powder and mix well.

PREPARATION OF WORKING SOLUTION

Phalloidin-iFluor™ 680 Conjugate working solution
Add 1 µL of Phalloidin-iFluor™ 680 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
  1. 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.
  2. Rinse the fixed cells 2–3 times in PBS.
  3. 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.
  4. Add 100 μL/well (96-well plate) of Phalloidin-iFluor™ 680 Conjugate working solution into the fixed cells, and stain the cells at room temperature for 20 to 90 minutes.
  5. Rinse cells gently with PBS 2 to 3 times to remove excess phalloidin conjugate before plating, sealing and imaging under microscope with Cy5.5 filter set. 

Spectrum

Product family

NameExcitation (nm)Emission (nm)Extinction coefficient (cm -1 M -1)Quantum yieldCorrection Factor (260 nm)Correction Factor (280 nm)
Phalloidin-iFluor® 350 Conjugate3454502000010.9510.830.23
Phalloidin-iFluor® 405 Conjugate4034273700010.9110.480.77
Phalloidin-iFluor® 488 Conjugate4915167500010.910.210.11
Phalloidin-iFluor® 514 Conjugate5115277500010.8310.2650.116
Phalloidin-iFluor® 532 Conjugate5375609000010.6810.260.16
Phalloidin-iFluor® 555 Conjugate55757010000010.6410.230.14
Phalloidin-iFluor® 594 Conjugate58760320000010.5310.050.04
Phalloidin-iFluor® 633 Conjugate64065425000010.2910.0620.044
Phalloidin-iFluor® 647 Conjugate65667025000010.2510.030.03
Phalloidin-iFluor® 700 Conjugate69071322000010.2310.090.04
Phalloidin-iFluor® 750 Conjugate75777927500010.1210.0440.039
Phalloidin-iFluor® 790 Conjugate78781225000010.1310.10.09
iFluor® 680-streptavidin conjugate68470122000010.2310.0970.094
Show More (4)

Citations

View all 33 citations: Citation Explorer
High-fat diet-induced LCN2 exacerbates myocardial ischemia-reperfusion injury by enhancing platelet activation
Authors: Li, Peng and Chen, Juhai and Wang, Mingdong and Wang, Qi and Liu, Xingde
Journal: Molecular Medicine Reports (2024): 1--9
Sinomenine increases osteogenesis in mice with ovariectomy-induced bone loss by modulating autophagy
Authors: Xiao, Hai-Xiang and Yu, Lei and Xia, Yu and Chen, Kai and Li, Wen-Ming and Ge, Gao-Ran and Zhang, Wei and Zhang, Qing and Zhang, Hong-Tao and Geng, De-Chun
Journal: World Journal of Stem Cells (2024): 486--498
Gut bacteria-derived membrane vesicles induce colonic dysplasia by inducing DNA damage in colon epithelial cells
Authors: Miyakawa, Yu and Otsuka, Motoyuki and Shibata, Chikako and Seimiya, Takahiro and Yamamoto, Keisuke and Ishibashi, Rei and Kishikawa, Takahiro and Tanaka, Eri and Isagawa, Takayuki and Takeda, Norihiko and others,
Journal: Cellular and Molecular Gastroenterology and Hepatology (2024)
Interfacing Perforated Eardrums with Graphene-Based Membranes for Broadband Hearing Recovery
Authors: Li, Chunyan and Xiong, Zhiyuan and Zhou, Lei and Huang, Weiluo and He, Yushi and Li, Linpeng and Shi, Haibo and Lu, Jiayu and Wang, Jian and Li, Dan and others,
Journal: Advanced Healthcare Materials (2022): 2201471
Calcium phosphate-adsorbable and acid-degradable carboxylated polyrotaxane consisting of $\beta$-cyclodextrins suppresses osteoclast resorptive activity
Authors: Yoshikawa, Yoshihiro and Tamura, Atsushi and Tsuda, Susumu and Domae, Eisuke and Zhang, Shunyao and Yui, Nobuhiko and Ikeo, Takashi and Yoshizawa, Tatsuya
Journal: Dental Materials Journal (2022): 2021--331

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
In vitro inhibition of OATP-mediated uptake of phalloidin using bile acid derivatives
Authors: Herraez E, Macias RI, Vazquez-Tato J, Vicens M, Monte MJ, Marin JJ.
Journal: Toxicol Appl Pharmacol (2009): 13
Page updated on October 9, 2024

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Physical properties

Molecular weight

~2000

Solvent

DMSO

Spectral properties

Correction Factor (260 nm)

0.097

Correction Factor (280 nm)

0.094

Extinction coefficient (cm -1 M -1)

2200001

Excitation (nm)

684

Emission (nm)

701

Quantum yield

0.231

Storage, safety and handling

H-phraseH301, H311, H331
Hazard symbolT
Intended useResearch Use Only (RUO)
R-phraseR23, R24, R25

Storage

Freeze (< -15 °C); Minimize light exposure
UNSPSC12352200
Fluorescence image of HeLa cells fixed with 4% formaldehyde then stained with Phalloidin-iFluor® 680 Conjugate (Cat#23128, Red) and nuclei stain Nuclear Green&trade; DCS1 (Cat#17550, Green)
Fluorescence image of HeLa cells fixed with 4% formaldehyde then stained with Phalloidin-iFluor® 680 Conjugate (Cat#23128, Red) and nuclei stain Nuclear Green&trade; DCS1 (Cat#17550, Green)
Fluorescence image of HeLa cells fixed with 4% formaldehyde then stained with Phalloidin-iFluor® 680 Conjugate (Cat#23128, Red) and nuclei stain Nuclear Green&trade; DCS1 (Cat#17550, Green)
Gallery Image 2