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AAT Bioquest

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
  1. Prepare samples in microplate wells
  2. Remove liquid from samples in the plate
  3. Add Phalloidin-iFluor™ 405 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 DAPI 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-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
  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™ 405 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 DAPI 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® 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® 680 Conjugate68470122000010.2310.0970.094
Phalloidin-iFluor® 700 Conjugate69071322000010.2310.090.04
Phalloidin-iFluor® 750 Conjugate75777927500010.1210.0440.039
Phalloidin-iFluor® 790 Conjugate78781225000010.1310.10.09
iFluor® 405-streptavidin conjugate4034273700010.9110.480.77
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
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
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)
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)
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

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

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Physical properties

Molecular weight

~1100

Solvent

DMSO

Spectral properties

Correction Factor (260 nm)

0.48

Correction Factor (280 nm)

0.77

Extinction coefficient (cm -1 M -1)

370001

Excitation (nm)

403

Emission (nm)

427

Quantum yield

0.911

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
Product Image
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Gallery Image 1
vLMW-F decrease actin polymerization and vesicle markers expression at plasma membrane. (A) LCLs (J1209, C0401, C1504) and DLBCLs (U2932, OCILy10, SUDHL4, SUDHL6) cells were treated or not (control) with 100µg/mL of native fucoidan or vLMW-F (F1 and F2) for 48 h, followed by phalloidin staining (F-actin, blue) and TOPRO-3 (nuclei, red) before confocal microscopy observations. Source: <b>Anti-Proliferative and Pro-Apoptotic vLMW Fucoidan Formulas Decrease PD-L1 Surface Expression in EBV Latency III and DLBCL Tumoral B-Cells by Decreasing Actin Network</b> by Saliba <em>et. al.</em>, <em>Mar. Drugs</em> Feb. 2023.
A Wnt ┫Pp1 signaling axis promotes ciliogenesis in motile cilia. Immunofluorescent image of MCCs for indicated proteins in St. 27 ccny/l1 morphants co-injected with Flag-ppp1r11 DNA. Actin labeled with Phalloidin-iFluor 405. Scale bar 5 µm. Source: <b>Mucociliary Wnt signaling promotes cilia biogenesis and beating</b> by Seidl <em>et. al.</em> <em>Nature Communications</em>. March 2023.