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Phalloidin-iFluor® 405 Conjugate

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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.
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Physical properties
Molecular weight~1100
SolventDMSO
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 yield0.911
Storage, safety and handling
H-phraseH301, H311, H331
Hazard symbolT
Intended useResearch Use Only (RUO)
R-phraseR23, R24, R25
StorageFreeze (< -15 °C); Minimize light exposure
UNSPSC12352200

OverviewpdfSDSpdfProtocol


Molecular weight
~1100
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
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


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spectrum

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 yield0.911

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)

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Citations


View all 52 citations: Citation Explorer
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)
Loss of polarity regulators initiates gasdermin-E-mediated pyroptosis in syncytiotrophoblasts
Authors: Patel, Khushali and Nguyen, Jasmine and Shaha, Sumaiyah and Brightwell, Amy and Duan, Wendy and Zubkowski, Ashley and Domingo, Ivan K and Riddell, Meghan
Journal: Life Science Alliance (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
Mucociliary Wnt signaling promotes cilia biogenesis and beating
Authors: Seidl, Carina and Da Silva, Fabio and Zhang, Kaiqing and Wohlgemuth, Kai and Omran, Heymut and Niehrs, Christof
Journal: Nature Communications (2023): 1259
Siglec-6 mediates the uptake of extracellular vesicles through a noncanonical glycolipid binding pocket
Authors: Schmidt, Edward N and Lamprinaki, Dimitra and McCord, Kelli A and Joe, Maju and Sojitra, Mirat and Waldow, Ayk and Nguyen, Jasmine and Monyror, John and Kitova, Elena N and Mozaneh, Fahima and others,
Journal: Nature Communications (2023): 2327
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
IFITM1 inhibits trophoblast invasion and is induced in placentas associated with IFN-mediated pregnancy diseases
Authors: Degrelle, S{\'e}verine A and Buchrieser, Julian and Dupressoir, Anne and Porrot, Fran{\c{c}}oise and Loeuillet, Laurence and Schwartz, Olivier and Fournier, Thierry
Journal: iScience (2023)
Down Syndrome is Associated with Altered Frequency and Functioning of Tracheal Multiciliated Cells, and Response to Influenza Virus Infection
Authors: Thomas, Samantha N and Niemeyer, Brian F and Jimenez-Valdes, Rocio J and Kaiser, Alexander J and Espinosa, Joaquin M and Sullivan, Kelly D and Goodspeed, Andrew and Costello, James C and Alder, Jonathan K and Ca{\~n}as-Arranz, Rodrigo and others,
Journal: iScience (2023)
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
Authors: Saliba, Jennifer and Manseur, Chanez and Groult, Hugo and Akil, Hussein and Tannoury, Mona and Troutaud, Danielle and Maugard, Thierry and Feuillard, Jean and Arnaudin, Ingrid and Jayat-Vignoles, Chantal
Journal: Marine Drugs (2023): 132

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
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
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
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
Processing of the phalloidin proprotein by prolyl oligopeptidase from the mushroom Conocybe albipes
Authors: Luo H, Hallen-Adams HE, Walton JD.
Journal: J Biol Chem (2009): 18070
Pygmy squids and giant brains: mapping the complex cephalopod CNS by phalloidin staining of vibratome sections and whole-mount preparations
Authors: Wollesen T, Loesel R, Wanninger A.
Journal: J Neurosci Methods (2009): 63
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