Phalloidin-iFluor® 647 Conjugate
Overview | ![]() ![]() |
See also: Antibodies and Proteomics, Antibody and Protein Labeling, Bioconjugation, Cell Structures and Organelles, Actin, iFluor® Dyes and Kits, Phalloidin Conjugates
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
- 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
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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 yield | 0.251 |
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 | 588 | 604 | 1800001 | 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 |
Show More (15) |
Images
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).
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.
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.
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 71 citations: Citation Explorer
Tuning Myogenesis by Controlling Gelatin Hydrogel Properties through Hydrogen Peroxide-Mediated Cross-Linking and Degradation
Authors: Mubarok, Wildan and Elvitigala, Kelum Chamara Manoj Lakmal and Sakai, Shinji
Journal: Gels (2022): 387
Authors: Mubarok, Wildan and Elvitigala, Kelum Chamara Manoj Lakmal and Sakai, Shinji
Journal: Gels (2022): 387
MARCKSL1 interacted with F-actin to promote esophageal squamous cell carcinoma mobility by modulating the formation of invadopodia
Authors: Zhao, Yue and Xie, Xiufeng and Tian, Lusong and Liu, Fang and Sun, Yulin and Lu, Haizhen and Zhao, Xiaohang and Mao, Yousheng
Journal: Cancer Medicine (2022)
Authors: Zhao, Yue and Xie, Xiufeng and Tian, Lusong and Liu, Fang and Sun, Yulin and Lu, Haizhen and Zhao, Xiaohang and Mao, Yousheng
Journal: Cancer Medicine (2022)
TNF$\alpha$-induced IDH1 hyperacetylation reprograms redox homeostasis and promotes the chemotherapeutic sensitivity
Authors: Yang, Hao and Zhao, Xiaoping and Liu, Jianjun and Jin, Mingming and Liu, Xiyu and Yan, Jun and Yao, Xufeng and Mao, Xinyi and Li, Nan and Liang, Beibei and others,
Journal: Oncogene (2022): 1--14
Authors: Yang, Hao and Zhao, Xiaoping and Liu, Jianjun and Jin, Mingming and Liu, Xiyu and Yan, Jun and Yao, Xufeng and Mao, Xinyi and Li, Nan and Liang, Beibei and others,
Journal: Oncogene (2022): 1--14
Calcium phosphate-adsorbable and acid-degradable carboxylated polyrotaxane consisting of $\beta$-cyclodextrins suppresses osteoclast resorptive activity
Authors: Yoshikawa, Yoshihiro and Tamura, Atsushi and Tsuda, Susumu and Domae, Eisuke and Zhang, Shunyao and Yui, Nobuhiko and Ikeo, Takashi and Yoshizawa, Tatsuya
Journal: Dental Materials Journal (2022): 2021--331
Authors: Yoshikawa, Yoshihiro and Tamura, Atsushi and Tsuda, Susumu and Domae, Eisuke and Zhang, Shunyao and Yui, Nobuhiko and Ikeo, Takashi and Yoshizawa, Tatsuya
Journal: Dental Materials Journal (2022): 2021--331
Multi-omics analyses of the ulcerative colitis gut microbiome link Bacteroides vulgatus proteases with disease severity
Authors: Mills, Robert H and Dulai, Parambir S and V{\'a}zquez-Baeza, Yoshiki and Sauceda, Consuelo and Daniel, No{\"e}mie and Gerner, Romana R and Batachari, Lakshmi E and Malfavon, Mario and Zhu, Qiyun and Weldon, Kelly and others,
Journal: Nature Microbiology (2022): 262--276
Authors: Mills, Robert H and Dulai, Parambir S and V{\'a}zquez-Baeza, Yoshiki and Sauceda, Consuelo and Daniel, No{\"e}mie and Gerner, Romana R and Batachari, Lakshmi E and Malfavon, Mario and Zhu, Qiyun and Weldon, Kelly and others,
Journal: Nature Microbiology (2022): 262--276
Actin remodelling controls proteasome homeostasis upon stress
Authors: Williams, Thomas D and Cacioppo, Roberta and Agrotis, Alexander and Black, Ailsa and Zhou, Houjiang and Rousseau, Adrien
Journal: bioRxiv (2022)
Authors: Williams, Thomas D and Cacioppo, Roberta and Agrotis, Alexander and Black, Ailsa and Zhou, Houjiang and Rousseau, Adrien
Journal: bioRxiv (2022)
Bioactive Cellulose Acetate Electrospun Mats as Scaffolds for Bone Tissue Regeneration
Authors: Laboy-L{\'o}pez, Simara and M{\'e}ndez Fern{\'a}ndez, Pedro O and Padilla-Zayas, Jorge G and Nicolau, Eduardo
Journal: International journal of biomaterials (2022)
Authors: Laboy-L{\'o}pez, Simara and M{\'e}ndez Fern{\'a}ndez, Pedro O and Padilla-Zayas, Jorge G and Nicolau, Eduardo
Journal: International journal of biomaterials (2022)
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
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
Conditioned medium from BV2 microglial cells having polyleucine specifically alters startle response in mice
Authors: Owada, Ryuji and Kakuta, Yohei and Yoshida, Kosuke and Mitsui, Shinichi and Nakamura, Kazuhiro
Journal: Scientific reports (2022): 1--15
Authors: Owada, Ryuji and Kakuta, Yohei and Yoshida, Kosuke and Mitsui, Shinichi and Nakamura, Kazuhiro
Journal: Scientific reports (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
Authors: Lin, Ruizhe and Kipreos, Edward T and Zhu, Jie and Khang, Chang Hyun and Kner, Peter
Journal: Nature communications (2021): 1--14
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
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
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
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
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
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
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
Authors: Genikhovich G, Technau U.
Journal: Cold Spring Harb Protoc (2009): pdb prot5283
AssayWise
Fluorescent Phalloidin: A Practical Stain for Visualizing Actin Filaments
Fluorescent Phalloidin: A Practical Stain for Visualizing Actin Filaments
Fluorescent Phalloidin: A Practical Stain for Visualizing Actin Filaments
Fluorescent Phalloidin: A Practical Stain for Visualizing Actin Filaments
Fluorescent Phalloidin: A Practical Stain for Visualizing Actin Filaments