Phalloidin-iFluor® 633 Conjugate

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Fax	1-800-609-2943
Email	sales@aatbio.com
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Physical properties

Molecular weight	1703.99
Solvent	DMSO

Spectral properties

Correction Factor (260 nm)	0.062
Correction Factor (280 nm)	0.044
Extinction coefficient (cm ^-1 M ^-1)	250000¹
Excitation (nm)	640
Emission (nm)	654
Quantum yield	0.29¹

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

1703.99

Correction Factor (260 nm)

0.062

Correction Factor (280 nm)

0.044

Extinction coefficient (cm ^-1 M ^-1)

250000¹

Excitation (nm)

640

Emission (nm)

654

Quantum yield

0.29¹

This deep red fluorescent phalloidin conjugate (equivalent to Alexa Fluor® 633-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™ 633 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™ 633 Conjugate stock solution

Add 30 µL of DMSO into the powder and mix well.

PREPARATION OF WORKING SOLUTION

Phalloidin-iFluor™ 633 Conjugate working solution

Add 1 µL of Phalloidin-iFluor™ 633 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™ 633 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® 633 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	58.686 µL	293.429 µL	586.858 µL	2.934 mL	5.869 mL
5 mM	11.737 µL	58.686 µL	117.372 µL	586.858 µL	1.174 mL
10 mM	5.869 µL	29.343 µL	58.686 µL	293.429 µL	586.858 µL

Molarity calculator

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

Mass (Calculate)		Molecular weight		Volume (Calculate)		Concentration (Calculate)		Moles
	/		=		x		=

Spectrum

Open in Advanced Spectrum Viewer

Spectral properties

Correction Factor (260 nm)	0.062
Correction Factor (280 nm)	0.044
Extinction coefficient (cm ^-1 M ^-1)	250000¹
Excitation (nm)	640
Emission (nm)	654
Quantum yield	0.29¹

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® 647 Conjugate	656	670	250000¹	0.25¹	0.03	0.03
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® 633-streptavidin conjugate	640	654	250000¹	0.29¹	0.062	0.044
Show More (4)

Images

Figure 1. Fluorescence images of HeLa cells stained with Phalloidin-iFluor® 633 Conjugate using fluorescence microscope with a Cy5 filter set. Live cells were stained with mitochondria dye MitoLite™ Green (Cat#22666, Green) and imaged. After fixation in 4% formaldehyde, the cells were labeled with F-actin dye iFluor® 633-Phalloidin (Red) and counterstained with Nuclear Blue™ DCS1 (Cat#17548, Blue).

Figure 2. Chemical structure for Phalloidin-iFluor® 633 Conjugate

Citations

View all 36 citations: Citation Explorer

Regulation of ACE2 isoforms by type 2 inflammation and viral infection in human airway epithelium
Authors: Stocker, N and Radzikowska, U and Wawrzyniak, P and Tan, G and Huang, M and Ding, M and Akdis, CA and Sokolowska, M
Journal: Mucosal Immunology (2023)

Evaluation of rapid transepithelial electrical resistance (TEER) measurement as a metric of kidney toxicity in a high-throughput microfluidic culture system
Authors: Shaughnessey, Erin M and Kann, Samuel H and Azizgolshani, Hesham and Black, Lauren D and Charest, Joseph L and Vedula, Else M
Journal: Scientific Reports (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

Synthetic Collagen-like Polymer That Undergoes a Sol--Gel Transition Triggered by O--N Acyl Migration at Physiological pH
Authors: Ichise, Shinichiro F and Koide, Takaki
Journal: International Journal of Molecular Sciences (2022): 1584

Three-dimensional characterization of collagen remodeling in cell-seeded collagen scaffolds via polarization second harmonic generation
Authors: Xydias, Dionysios and Ziakas, Georgios and Psilodimitrakopoulos, Sotiris and Lemonis, Andreas and Bagli, Eleni and Fotsis, Theodore and Gravanis, Achille and Tzeranis, Dimitrios S and Stratakis, Emmanuel
Journal: Biomedical optics express (2021): 1136--1153

Proteogenomic analysis of Inhibitor of Differentiation 4 (ID4) in basal-like breast cancer
Authors: Swarbrick, A and Baker, LA and Holliday, H and Roden, D and Krisp, C and Wu, SZ and Junankar, S and Serandour, AA and Mohammed, H and Nair, R and others,
Journal: (2020)

Using enhanced Green fluorescence protein-expressing Escherichia Coli to assess mouse peritoneal macrophage phagocytosis
Authors: Zhang, Yu and Wang, Guan and Lu, Jia and Xu, Li-ming and Xiong, Jun-yu
Journal: JoVE (Journal of Visualized Experiments) (2019): e58751

Cigarette smoking promotes cancer-related transformation of oral epithelial cells through activation of Wnt and MAPK pathway
Authors: Sarkar, Ripon and Das, Ankita and Paul, Ranjan R and Barui, Ananya
Journal: Future Oncology (2019)

Enhanced bovine serum albumin absorption on the N-hydroxysuccinimide activated graphene oxide and its corresponding cell affinity
Authors: Xiong, Kun and Fan, Qingbo and Wu, Tingting and Shi, Haishan and Chen, Lin and Yan, Minhao
Journal: Materials Science and Engineering: C (2017)

Cell-Permeable, MMP-2 Activatable, Nickel Ferrite and His-tagged Fusion Protein Self-Assembled Fluorescent Nanoprobe for Tumor Magnetic Targeting and Imaging
Authors: Sun, Lu and Xie, Shuping and Qi, Jing and Liu, Ergang and Liu, Di and Liu, Quan and Chen, Sunhui and He, Huining and Yang, Victor C
Journal: ACS Applied Materials & Interfaces (2017)

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