Phalloidin Conjugates
This yellow fluorescent phalloidin conjugate (equivalent to Alexa Fluor® 514-labeled phalloidin) selectively binds to F-actins. Phalloidin derivatives are convenient probes used at nanomolar concentrations 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 and 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 valuable 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 been enormously helpful in localizing actin filaments in living or fixed cells and visualizing individual actin filaments in vitro. Fluorescent phalloidin derivatives have been used as an important tool in studying 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-iFluor™ 514 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 TRITC filter
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-iFluor™ 514 Conjugate working solution
Add 1 µL of Phalloidin-iFluor™ 514 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-iFluor™ 514 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 TRITC filter set.
Spectrum
Open in Advanced Spectrum Viewer
Alternative formats
Name | Conjugate |
Phalloidin-iFluor® 488 Conjugate | iFluor 488 |
Phalloidin-iFluor® 647 Conjugate | iFluor 647 |
Phalloidin-iFluor® 594 Conjugate | iFluor 594 |
Phalloidin-iFluor® 555 Conjugate | iFluor 555 |
Phalloidin-iFluor® 633 Conjugate | iFluor 633 |
Phalloidin-iFluor® 532 Conjugate | iFluor 532 |
Phalloidin-iFluor® 405 Conjugate | iFluor 405 |
Phalloidin-iFluor® 514 Conjugate | iFluor 514 |
Phalloidin-iFluor® 350 Conjugate | iFluor 350 |
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Product family
Name | Excitation (nm) | Emission (nm) | Extinction coefficient (cm -1 M -1) | Quantum yield | Correction Factor (260 nm) | Correction Factor (280 nm) |
Phalloidin-iFluor® 350 Conjugate | 345 | 450 | 200001 | 0.951 | 0.83 | 0.23 |
Phalloidin-iFluor® 405 Conjugate | 403 | 427 | 370001 | 0.911 | 0.48 | 0.77 |
Phalloidin-iFluor® 488 Conjugate | 491 | 516 | 750001 | 0.91 | 0.21 | 0.11 |
Phalloidin-iFluor® 532 Conjugate | 537 | 560 | 900001 | 0.681 | 0.26 | 0.16 |
Phalloidin-iFluor® 555 Conjugate | 557 | 570 | 1000001 | 0.641 | 0.23 | 0.14 |
Phalloidin-iFluor® 594 Conjugate | 587 | 603 | 2000001 | 0.531 | 0.05 | 0.04 |
Phalloidin-iFluor® 633 Conjugate | 640 | 654 | 2500001 | 0.291 | 0.062 | 0.044 |
Phalloidin-iFluor® 647 Conjugate | 656 | 670 | 2500001 | 0.251 | 0.03 | 0.03 |
Phalloidin-iFluor® 680 Conjugate | 684 | 701 | 2200001 | 0.231 | 0.097 | 0.094 |
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Citations
View all 39 citations: Citation Explorer
Isolated Botanical Polyphenol Actin-Binding Compounds Impact Human Dermal Fibroblast Morphology: Potential Anti-Aging Effects and Insight Into YAP/TAZ Mechanical Cytoskeletal Modulation
Authors: Elia, Marina
Journal: (2024)
Authors: Elia, Marina
Journal: (2024)
Influences of topography and composition on hepatocytes within electrospun polycaprolactone scaffolds for liver tissue engineering
Authors: Gao, Yunxi
Journal: (2023)
Authors: Gao, Yunxi
Journal: (2023)
Atomic force microscopy--based assessment of multimechanical cellular properties for classification of graded bladder cancer cells and cancer early diagnosis using machine learning analysis
Authors: Zhu, Xinyao and Qin, Rui and Qu, Kaige and Wang, Zuobin and Zhao, Xuexia and Xu, Wei
Journal: Acta Biomaterialia (2023): 358--373
Authors: Zhu, Xinyao and Qin, Rui and Qu, Kaige and Wang, Zuobin and Zhao, Xuexia and Xu, Wei
Journal: Acta Biomaterialia (2023): 358--373
In situ FRET-based localization of the N-terminus of myosin binding protein-C in heart muscle cells
Authors: Chandler, Jessica and Treacy, Conor and Ameer-Beg, Simon and Ehler, Elisabeth and Irving, Malcolm and Kampourakis, Thomas
Journal: Proceedings of the National Academy of Sciences of the United States of America (2023)
Authors: Chandler, Jessica and Treacy, Conor and Ameer-Beg, Simon and Ehler, Elisabeth and Irving, Malcolm and Kampourakis, Thomas
Journal: Proceedings of the National Academy of Sciences of the United States of America (2023)
Hypothiocyanous Acid Disrupts the Barrier Function of Brain Endothelial Cells
Authors: van Leeuwen, Eveline and Hampton, Mark B and Smyth, Leon CD
Journal: Antioxidants (2022): 608
Authors: van Leeuwen, Eveline and Hampton, Mark B and Smyth, Leon CD
Journal: Antioxidants (2022): 608
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
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
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
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
Authors: Chazotte B., undefined
Journal: Cold Spring Harb Protoc (2010): pdb prot4947
Protective effect of bile acid derivatives in phalloidin-induced rat liver toxicity
Authors: Herraez E, Macias RI, Vazquez-Tato J, Hierro C, Monte MJ, Marin JJ.
Journal: Toxicol Appl Pharmacol (2009): 21
Authors: Herraez E, Macias RI, Vazquez-Tato J, Hierro C, Monte MJ, Marin JJ.
Journal: Toxicol Appl Pharmacol (2009): 21
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