Phalloidin Conjugates
This blue fluorescent phalloidin conjugate (equivalent to Alexa Fluor® 405-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™ 405 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 DAPI 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™ 405 Conjugate working solution
Add 1 µL of Phalloidin-iFluor™ 405 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™ 405 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 DAPI 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® 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® 647 Conjugate | 656 | 670 | 2500001 | 0.251 | 0.03 | 0.03 |
Phalloidin-iFluor® 680 Conjugate | 684 | 701 | 2200001 | 0.231 | 0.097 | 0.094 |
Show More (4) |
Citations
View all 55 citations: Citation Explorer
Hippo signaling pathway regulates Ebola virus transcription and egress
Authors: Liang, Jingjing and Djurkovic, Marija A and Leavitt, Carson G and Shtanko, Olena and Harty, Ronald N
Journal: Nature Communications (2024): 6953
Authors: Liang, Jingjing and Djurkovic, Marija A and Leavitt, Carson G and Shtanko, Olena and Harty, Ronald N
Journal: Nature Communications (2024): 6953
A novel delta opioid receptor specific peptide reduces craving in an animal model of cocaine seeking
Authors: Itzhak, Pnina Shirel and Yael, Hevroni and Matsree, Erez and Pee'r-Nissan, Hilla and Lancman, Shira Ofer and Barnea, R and Luboshits, G and Motiei, Menachem and Betzer, Oshra and Gispan, Iris and others,
Journal: Addiction Neuroscience (2024): 100159
Authors: Itzhak, Pnina Shirel and Yael, Hevroni and Matsree, Erez and Pee'r-Nissan, Hilla and Lancman, Shira Ofer and Barnea, R and Luboshits, G and Motiei, Menachem and Betzer, Oshra and Gispan, Iris and others,
Journal: Addiction Neuroscience (2024): 100159
Osteoblast cell behavior on polyetheretherketone dental implant surfaces treated with different grit size aluminum oxide particles: An in vitro analysis
Authors: Gaikwad, Amit and Parizi, Marjan Kheirmand and Winkel, Andreas and Stiesch, Meike
Journal: The Journal of Prosthetic Dentistry (2024)
Authors: Gaikwad, Amit and Parizi, Marjan Kheirmand and Winkel, Andreas and Stiesch, Meike
Journal: The Journal of Prosthetic Dentistry (2024)
mRNA-Laden Lipid-Nanoparticle-Enabled in Situ CAR-Macrophage Engineering for the Eradication of Multidrug-Resistant Bacteria in a Sepsis Mouse Model
Authors: Tang, Chunwei and Jing, Weiqiang and Han, Kun and Yang, Zhenmei and Zhang, Shengchang and Liu, Miaoyan and Zhang, Jing and Zhao, Xiaotian and Liu, Ying and Shi, Chongdeng and others,
Journal: ACS nano (2024)
Authors: Tang, Chunwei and Jing, Weiqiang and Han, Kun and Yang, Zhenmei and Zhang, Shengchang and Liu, Miaoyan and Zhang, Jing and Zhao, Xiaotian and Liu, Ying and Shi, Chongdeng and others,
Journal: ACS nano (2024)
Repurposing tamoxifen as potential host-directed therapeutic for tuberculosis
Authors: Boland, Ralf and Heemskerk, Matthias T and Forn-Cun{\'\i}, Gabriel and Korbee, Cornelis J and Walburg, Kimberley V and Esselink, Jeroen J and Carvalho dos Santos, Carina and de Waal, Amy M and van der Hoeven, Daniel CM and van der Sar, Elisa and others,
Journal: Mbio (2023): e03024--22
Authors: Boland, Ralf and Heemskerk, Matthias T and Forn-Cun{\'\i}, Gabriel and Korbee, Cornelis J and Walburg, Kimberley V and Esselink, Jeroen J and Carvalho dos Santos, Carina and de Waal, Amy M and van der Hoeven, Daniel CM and van der Sar, Elisa and others,
Journal: Mbio (2023): e03024--22
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
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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.
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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
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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
Effect of Phalloidin on Filaments Polymerized from Heart Muscle Adp-Actin Monomers
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Journal: J Therm Anal Calorim (2009): 721
Authors: Vig A, Dudas R, Kupi T, Orban J, Hild G, Lorinczy D, Nyitrai M.
Journal: J Therm Anal Calorim (2009): 721
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