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Phalloidin Conjugates
This deep red fluorescent phalloidin conjugate (equivalent to Alexa Fluor® 647-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-iFluor™ 647 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 Cy5 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 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™ 647 Conjugate stock solution
Add 30 µL of DMSO into the powder and mix well.PREPARATION OF WORKING SOLUTION
Phalloidin-iFluor™ 647 Conjugate working solution
Add 1 µL of Phalloidin-iFluor™ 647 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™ 647 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 Cy5 filter set.
Calculators
Common stock solution preparation
Table 1. Volume of DMSO needed to reconstitute specific mass of Phalloidin-iFluor® 647 Conjugate to given concentration. Note that volume is only for preparing stock solution. Refer to sample experimental protocol for appropriate experimental/physiological buffers.
| 0.1 mg | 0.5 mg | 1 mg | 5 mg | 10 mg | |
| 1 mM | 70.99 µL | 354.95 µL | 709.9 µL | 3.549 mL | 7.099 mL |
| 5 mM | 14.198 µL | 70.99 µL | 141.98 µL | 709.9 µL | 1.42 mL |
| 10 mM | 7.099 µL | 35.495 µL | 70.99 µL | 354.95 µL | 709.9 µL |
Molarity calculator
Enter any two values (mass, volume, concentration) to calculate the third.
| Mass (Calculate) | Molecular weight | Volume (Calculate) | Concentration (Calculate) | Moles | ||||
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Spectrum
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® 514 Conjugate | 511 | 527 | 750001 | 0.831 | 0.265 | 0.116 |
| 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® 680 Conjugate | 684 | 701 | 2200001 | 0.231 | 0.097 | 0.094 |
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Citations
View all 138 citations: Citation Explorer
Shrinkable Hydrogels through Host--Guest Interactions: A Robust Approach to Obtain Tubular Cell-Laden Scaffolds with Small Diameters
Authors: Iudin, Dmitrii and van Steenbergen, Mies J and Masereeuw, Rosalinde and van Ravensteijn, Bas GP and Vermonden, Tina
Journal: Advanced Functional Materials (2024): 2416522
Authors: Iudin, Dmitrii and van Steenbergen, Mies J and Masereeuw, Rosalinde and van Ravensteijn, Bas GP and Vermonden, Tina
Journal: Advanced Functional Materials (2024): 2416522
Fe3O4 nanoparticles containing gambogic acid inhibit metastasis in colorectal cancer via the RORB/EMILIN1 axis
Authors: Fan, Xiaodong and Lv, Chunyang and Xue, Meiling and Meng, Peng and Qian, Xiaoping
Journal: Cell Adhesion \& Migration (2024): 38--53
Authors: Fan, Xiaodong and Lv, Chunyang and Xue, Meiling and Meng, Peng and Qian, Xiaoping
Journal: Cell Adhesion \& Migration (2024): 38--53
Colorectal carcinoma progression is not influenced by the pseudokinase PEAK1
Authors: Zuidema, Alba and Atherton, Paul and van der Poel, Sabine and Kreft, Maaike and Song, Ji-Ying and Bierbooms, Martine and Verhoeven, Sophie and Papagianni, Chrysoula and Kroese, Lona and Ali, Rahmen Bin and others,
Journal: Scientific Reports (2024): 27663
Authors: Zuidema, Alba and Atherton, Paul and van der Poel, Sabine and Kreft, Maaike and Song, Ji-Ying and Bierbooms, Martine and Verhoeven, Sophie and Papagianni, Chrysoula and Kroese, Lona and Ali, Rahmen Bin and others,
Journal: Scientific Reports (2024): 27663
Carbon quantum dots in breast cancer modulate cellular migration via cytoskeletal and nuclear structure
Authors: Dinger, Nikita and Russo, Carmela and Fusco, Sabato and Netti, Paolo A and Sirignano, Mariano and Panzetta, Valeria
Journal: Nanotoxicology (2024): 1--27
Authors: Dinger, Nikita and Russo, Carmela and Fusco, Sabato and Netti, Paolo A and Sirignano, Mariano and Panzetta, Valeria
Journal: Nanotoxicology (2024): 1--27
Mechanical Stimulation and Aligned Poly ($\varepsilon$-caprolactone)--Gelatin Electrospun Scaffolds Promote Skeletal Muscle Regeneration
Authors: Calero-Castro, Francisco Jos{\'e} and Perez-Puyana, V{\'\i}ctor Manuel and Laga, Im{\'a}n and Padillo Ruiz, Javier and Romero, Alberto and de la Portilla de Juan, Fernando
Journal: ACS Applied Bio Materials (2024)
Authors: Calero-Castro, Francisco Jos{\'e} and Perez-Puyana, V{\'\i}ctor Manuel and Laga, Im{\'a}n and Padillo Ruiz, Javier and Romero, Alberto and de la Portilla de Juan, Fernando
Journal: ACS Applied Bio Materials (2024)
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
Anti-acetylated tubulin antibody staining and phalloidin staining in the starlet sea anemone Nematostella vectensis
Authors: Genikhovich G, Technau U.
Journal: Cold Spring Harb Protoc (2009): pdb prot5283
Authors: Genikhovich G, Technau U.
Journal: Cold Spring Harb Protoc (2009): pdb prot5283
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