Phalloidin-iFluor® 647 Conjugate

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Physical properties

Molecular weight	1408.65
Solvent	DMSO

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

Storage, safety and handling

Certificate of Origin	Download PDF
H-phrase	H301, H311, H331
Hazard symbol	T
Intended use	Research Use Only (RUO)
R-phrase	R23, R24, R25
Storage	Freeze (< -15 °C); Minimize light exposure
UNSPSC	12352200

Overview SDS Protocol

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

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

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

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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
Phalloidin-iFluor® 700 Conjugate	690	713	220000¹	0.23¹	0.09	0.04
Phalloidin-iFluor® 750 Conjugate	757	779	275000¹	0.12¹	0.044	0.039
Phalloidin-iFluor® 790 Conjugate	787	812	250000¹	0.13¹	0.1	0.09
iFluor® 647-streptavidin conjugate	656	670	250000¹	0.25¹	0.03	0.03
Cholyl-iFluor® 647 conjugate	656	670	250000¹	0.25¹	0.03	0.03
Show More (5)

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 4. Chemical structure for Phalloidin-iFluor® 647 Conjugate

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.

Co-localization of caveolin-1 and actin in post-conditioned hearts treated with latrunculin A. Representative confocal laser microscopy of the sections of frozen heart tissue after double-immunofluorescence staining of caveolin-1 (green) and actin (red). Cell nuclei were counterstained with DAPI. Source: <b>Actin-Cytoskeleton Drives Caveolae Signaling to Mitochondria during Postconditioning</b> by Correa <em>et. al.</em>, <em>Cells</em>. Feb. 2023.

Figure 6. Co-localization of caveolin-1 and actin in post-conditioned hearts treated with latrunculin A. Representative confocal laser microscopy of the sections of frozen heart tissue after double-immunofluorescence staining of caveolin-1 (green) and actin (red). Cell nuclei were counterstained with DAPI. Source: Actin-Cytoskeleton Drives Caveolae Signaling to Mitochondria during Postconditioning by Correa et. al., Cells. Feb. 2023.

Cytokines induce intracellular component and cytoskeleton changes in RA synoviocytes. Holographic and tomographic microscopy images and phalloidin staining. Phalloidin-iFluor 647 was used for staining. Source: <b>Effects of pro-inflammatory cytokines and cell interactions on cell area and cytoskeleton of rheumatoid arthritis synoviocytes and immune cells</b> by Filali <em>et. al.</em>, <em>European Journal of Cell Biology</em>. March 2023

Figure 7. Cytokines induce intracellular component and cytoskeleton changes in RA synoviocytes. Holographic and tomographic microscopy images and phalloidin staining. Phalloidin-iFluor 647 was used for staining. Source: Effects of pro-inflammatory cytokines and cell interactions on cell area and cytoskeleton of rheumatoid arthritis synoviocytes and immune cells by Filali et. al., European Journal of Cell Biology. March 2023

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References

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Improved penile histology by phalloidin stain: circular and longitudinal cavernous smooth muscles, dual-endothelium arteries, and erectile dysfunction-associated changes
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Journal: Urology (2011): 970 e1

Phalloidin perturbs the interaction of human non-muscle myosin isoforms 2A and 2C1 with F-actin
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Journal: FEBS Lett (2011): 767

pH-(low)-insertion-peptide (pHLIP) translocation of membrane impermeable phalloidin toxin inhibits cancer cell proliferation
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Labeling cytoskeletal F-actin with rhodamine phalloidin or fluorescein phalloidin for imaging
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Effect of Phalloidin on Filaments Polymerized from Heart Muscle Adp-Actin Monomers
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Pygmy squids and giant brains: mapping the complex cephalopod CNS by phalloidin staining of vibratome sections and whole-mount preparations
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Anti-acetylated tubulin antibody staining and phalloidin staining in the starlet sea anemone Nematostella vectensis
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