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Phalloidin-iFluor™ 647 Conjugate

Fluorescence images of HeLa cells stained with Phalloidin-iFluor™ 647 Conjugate using fluorescence microscope with a Cy5 filter set (Red). Live cells were first stained with mitochondria dye MitoLite™ Green. After fixation in 4% formaldehyde, cells were labeled with Phalloidin-iFluor™ 647 and counterstained with Nuclear Blue™ DCS1 (Cat#17548, Blue).
Fluorescence images of HeLa cells stained with Phalloidin-iFluor™ 647 Conjugate using fluorescence microscope with a Cy5 filter set (Red). Live cells were first stained with mitochondria dye MitoLite™ Green. After fixation in 4% formaldehyde, cells were labeled with Phalloidin-iFluor™ 647 and counterstained with Nuclear Blue™ DCS1 (Cat#17548, Blue).
VG and IF images of the ORL. The periosteal (row A), intramuscular (row B), preorbicularis (row C) and dermal (row D) regions of the ORL were observed by VG (column 1) and IF (columns 2–5). There were immunopositive reactions for elastin (column 2, blue), collagen type I (column 3, green) and actin (column 4, red). The column 5 shows a merged image of the images in columns 2–4 image. Asterisks indicate confluence of the perimysium into the ORL fibres. Source: <strong>Three-dimensional structure of the orbicularis retaining ligament: an anatomical study using micro-computed tomography</strong> by Jehoon O et al., <em>Scientific Reports</em>, Nov. 2018.
Overall structure of the orbicularis retaining ligament (ORL). (a) Three-dimensional (3D) morphology reconstructed from micro-computed tomography (mCT) image sections. (b) Modified Verhoeff Van Gieson staining (VG) image. (c) A merged immunofluorescence (IF) image (elastin, blue; collagen type I, green; actin, red). Arrowheads indicate a direct fibre from the periosteum (P) to the dermis (D). OOc, orbicularis oculi muscle. S, sagittal; M, medial; A, anterior. Source: <strong>Three-dimensional structure of the orbicularis retaining ligament: an anatomical study using micro-computed tomography</strong> by Jehoon O et al., <em>Scientific Reports</em>, Nov. 2018. 
Chemical structure for Phalloidin-iFluor™ 647 Conjugate
Fluorescence images of HeLa cells stained with Phalloidin-iFluor™ 647 Conjugate using fluorescence microscope with a Cy5 filter set (Red). HeLa cells were fixed with 4% formaldehyde followed by incubation with 1 ug/mL mouse tubulin antibody. Cells were stained with 10 ug/mL of GxM IgG- iFluor 488 conjugates. Cells were stained with Phalloidin-iFluor™ 647 conjugate following product protocol and incubated with 2 uM DAPI for 5 min before imaging.
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Catalog Number23127
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Telephone1-408-733-1055
Fax1-408-733-1304
Emailsales@aatbio.com
InternationalSee distributors
ShippingStandard overnight for United States, inquire for international
Physical properties
Molecular weight1408.65
SolventDMSO
Spectral properties
Correction Factor (260 nm)0.03
Correction Factor (280 nm)0.03
Correction Factor (656 nm)0.0793
Extinction coefficient (cm -1 M -1)2500001
Excitation (nm)656
Emission (nm)670
Quantum yield0.251
Storage, safety and handling
Certificate of OriginDownload PDF
H-phraseH301, H311, H331
Hazard symbolT
Intended useResearch Use Only (RUO)
R-phraseR23, R24, R25
StorageFreeze (< -15 °C); Minimize light exposure
UNSPSC12352200

OverviewpdfSDSpdfProtocol


See also: Actin, Phalloidin Conjugates, Cell Structures and Organelles
Molecular weight
1408.65
Correction Factor (260 nm)
0.03
Correction Factor (280 nm)
0.03
Correction Factor (656 nm)
0.0793
Extinction coefficient (cm -1 M -1)
2500001
Excitation (nm)
656
Emission (nm)
670
Quantum yield
0.251
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
  1. Prepare samples in microplate wells
  2. Remove liquid from samples in the plate
  3. Add Phalloidin-iFluor™ 647 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 Cy5 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 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
  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™ 647 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 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 mg0.5 mg1 mg5 mg10 mg
1 mM70.99 µL354.95 µL709.9 µL3.549 mL7.099 mL
5 mM14.198 µL70.99 µL141.98 µL709.9 µL1.42 mL
10 mM7.099 µL35.495 µL70.99 µL354.95 µL709.9 µL

Molarity calculator

Enter any two values (mass, volume, concentration) to calculate the third.

Mass (Calculate)Molecular weightVolume (Calculate)Concentration (Calculate)Moles
/=x=

Spectrum


Open in Advanced Spectrum Viewer
spectrum

Spectral properties

Correction Factor (260 nm)0.03
Correction Factor (280 nm)0.03
Correction Factor (656 nm)0.0793
Extinction coefficient (cm -1 M -1)2500001
Excitation (nm)656
Emission (nm)670
Quantum yield0.251

Images


Fluorescence images of HeLa cells stained with Phalloidin-iFluor™ 647 Conjugate using fluorescence microscope with a Cy5 filter set (Red). Live cells were first stained with mitochondria dye MitoLite™ Green. After fixation in 4% formaldehyde, cells were labeled with Phalloidin-iFluor™ 647 and counterstained with Nuclear Blue™ DCS1 (Cat#17548, Blue).
Figure 1. Fluorescence images of HeLa cells stained with Phalloidin-iFluor™ 647 Conjugate using fluorescence microscope with a Cy5 filter set (Red). Live cells were first stained with mitochondria dye MitoLite™ Green. After fixation in 4% formaldehyde, cells were labeled with Phalloidin-iFluor™ 647 and counterstained with Nuclear Blue™ DCS1 (Cat#17548, Blue).
VG and IF images of the ORL. The periosteal (row A), intramuscular (row B), preorbicularis (row C) and dermal (row D) regions of the ORL were observed by VG (column 1) and IF (columns 2–5). There were immunopositive reactions for elastin (column 2, blue), collagen type I (column 3, green) and actin (column 4, red). The column 5 shows a merged image of the images in columns 2–4 image. Asterisks indicate confluence of the perimysium into the ORL fibres. Source: <strong>Three-dimensional structure of the orbicularis retaining ligament: an anatomical study using micro-computed tomography</strong> by Jehoon O et al., <em>Scientific Reports</em>, Nov. 2018.
Figure 2. VG and IF images of the ORL. The periosteal (row A), intramuscular (row B), preorbicularis (row C) and dermal (row D) regions of the ORL were observed by VG (column 1) and IF (columns 2–5). There were immunopositive reactions for elastin (column 2, blue), collagen type I (column 3, green) and actin (column 4, red). The column 5 shows a merged image of the images in columns 2–4 image. Asterisks indicate confluence of the perimysium into the ORL fibres. Source: Three-dimensional structure of the orbicularis retaining ligament: an anatomical study using micro-computed tomography by Jehoon O et al., Scientific Reports, Nov. 2018.
Overall structure of the orbicularis retaining ligament (ORL). (a) Three-dimensional (3D) morphology reconstructed from micro-computed tomography (mCT) image sections. (b) Modified Verhoeff Van Gieson staining (VG) image. (c) A merged immunofluorescence (IF) image (elastin, blue; collagen type I, green; actin, red). Arrowheads indicate a direct fibre from the periosteum (P) to the dermis (D). OOc, orbicularis oculi muscle. S, sagittal; M, medial; A, anterior. Source: <strong>Three-dimensional structure of the orbicularis retaining ligament: an anatomical study using micro-computed tomography</strong> by Jehoon O et al., <em>Scientific Reports</em>, Nov. 2018. 
Figure 3. Overall structure of the orbicularis retaining ligament (ORL). (a) Three-dimensional (3D) morphology reconstructed from micro-computed tomography (mCT) image sections. (b) Modified Verhoeff Van Gieson staining (VG) image. (c) A merged immunofluorescence (IF) image (elastin, blue; collagen type I, green; actin, red). Arrowheads indicate a direct fibre from the periosteum (P) to the dermis (D). OOc, orbicularis oculi muscle. S, sagittal; M, medial; A, anterior. Source: Three-dimensional structure of the orbicularis retaining ligament: an anatomical study using micro-computed tomography by Jehoon O et al., Scientific Reports, Nov. 2018. 
Chemical structure for Phalloidin-iFluor™ 647 Conjugate
Figure 4. Chemical structure for Phalloidin-iFluor™ 647 Conjugate
Fluorescence images of HeLa cells stained with Phalloidin-iFluor™ 647 Conjugate using fluorescence microscope with a Cy5 filter set (Red). HeLa cells were fixed with 4% formaldehyde followed by incubation with 1 ug/mL mouse tubulin antibody. Cells were stained with 10 ug/mL of GxM IgG- iFluor 488 conjugates. Cells were stained with Phalloidin-iFluor™ 647 conjugate following product protocol and incubated with 2 uM DAPI for 5 min before imaging.
Figure 5. Fluorescence images of HeLa cells stained with Phalloidin-iFluor™ 647 Conjugate using fluorescence microscope with a Cy5 filter set (Red). HeLa cells were fixed with 4% formaldehyde followed by incubation with 1 ug/mL mouse tubulin antibody. Cells were stained with 10 ug/mL of GxM IgG- iFluor 488 conjugates. Cells were stained with Phalloidin-iFluor™ 647 conjugate following product protocol and incubated with 2 uM DAPI for 5 min before imaging.

Citations


View all 62 citations: Citation Explorer
Autophagy facilitates age-related cell apoptosis—a new insight from senile cataract
Authors: Huang, Jiani and Yu, Wangshu and He, Qin and He, Xiaoying and Yang, Ming and Chen, Wei and Han, Wei
Journal: Cell death \& disease (2022): 1--15
Subcellular three-dimensional imaging deep through multicellular thick samples by structured illumination microscopy and adaptive optics
Authors: Lin, Ruizhe and Kipreos, Edward T and Zhu, Jie and Khang, Chang Hyun and Kner, Peter
Journal: Nature communications (2021): 1--14
Microfluidic Co-Culture Platform to Recapitulate the Maternal--Placental--Embryonic Axis
Authors: Boos, Julia A and Misun, Patrick M and Brunoldi, Giulia and Furer, Lea A and Aengenheister, Leonie and Modena, Mario and Rousset, Nassim and Buerki-Thurnherr, Tina and Hierlemann, Andreas
Journal: Advanced Biology (2021): 2100609
Piezo1 channels contribute to the regulation of human atrial fibroblast mechanical properties and matrix stiffness sensing
Authors: Emig, Ramona and Knodt, Wiebke and Krussig, Mario J and Zgierski-Johnston, Callum M and Gorka, Oliver and Gro{\ss}, Olaf and Kohl, Peter and Ravens, Ursula and Peyronnet, R{\'e}mi
Journal: Cells (2021): 663
A novel adhesive complex at the base of intestinal microcilli
Authors: Ebnet, Klaus T and Hartmann, Christian and Th{\"u}ring, E and Michels, Brigitta E and Pajonczyk, Denise and Leu{\ss}ink, Sophia and Greune, Lilo and Brinkmann, Frauke and Glaesner-Ebnet, Mark and Wardelmann, Eva and others,
Journal: (2021)
Niche-guided tissue patterning by chemomechanical flow lithography
Authors: Newman, Peter LH and Osteil, Pierre and Anderson, Timothy A and Sun, Jane QJ and Kempe, Daryan and Biro, Mat{\'e} and Tam, Patrick PL and Shin, Jae-Won and Zreiqat, Hala
Journal: bioRxiv (2021)
In Vitro-Evolved Peptides Mimic a Binding Motif of the G-Actin-Binding Protein Thymosin-B4 and Serve as Research Tools
Authors: G{\"u}beli, Raphael and Bertoldo, Davide and Shimada, Kenji and Gerhold, Christian and Hurst, Verena and Takahashi, Yuichiro and Wilbs, Jonas and Harata, Masahiko and M Gasser, Susan and Heinis, Christian
Journal: (2020)
Continuous chaotic bioprinting of skeletal muscle-like constructs
Authors: Bol{\'\i}var-Monsalve, Edna Johana and Ceballos-Gonz{\'a}lez, Carlos Fernando and Borrayo-Monta{\~n}o, Karen Ixchel and Quevedo-Moreno, Diego Alonso and Yee-de Le{\'o}n, Juan Felipe and Khademhosseini, Ali and Weiss, Paul S and Alvarez, Mario Mois{\'e}s and Trujillo-de Santiago, Grissel
Journal: Bioprinting (2020): e00125

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

Documents


Certificate of origin
Safety data sheet
Product protocol
Certificate of analysis

FAQ


Can we fix cells with glutaraldehyde and then stain with fluorescent phalloidin?
How does phalloidin affect actin assembly?
Is phalloidin cell permeable?
What does rhodamine phalloidin do?
When using Cell Navigator™ Fluorimetric Lipid Droplet Assay Kit which fluorescent dye should I use?
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