XFD488 Phalloidin XFD488 Same Structure to Alexa Fluor™ 488

<strong>Fixed and stained HeLa cells.</strong><br>HeLa cells were fixed with 4% formaldehyde, permeabilized, and blocked. F-actin were stained with XFD488 phalloidin (Cat No. 23153) and nuclei labeled with Nuclear Red™ DCS1 (Cat No. 17552). Images were acquired on a Keyence BZ-X710 all-in-one fluorescence microscope.

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Physical properties

Molecular weight	~1300
Solvent	DMSO

Spectral properties

Correction Factor (260 nm)	0.3
Correction Factor (280 nm)	0.11
Extinction coefficient (cm ^-1 M ^-1)	73000
Excitation (nm)	499
Emission (nm)	520
Quantum yield	0.92¹

Storage, safety and handling

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

~1300

Correction Factor (260 nm)

0.3

Correction Factor (280 nm)

0.11

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

73000

Excitation (nm)

499

Emission (nm)

520

Quantum yield

0.92¹

XFD488 is manufactured by AAT Bioquest, and it has the same chemical structure of Alexa Fluor® 488 (Alexa Fluor® is the trademark of ThermoFisher). XFD488 phalloidin conjugate is chemically equivalent to Alexa Fluor® 488 phalloidin. This green fluorescent phalloidin conjugate 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. 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 XFD488 Phalloidin 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 FITC 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

XFD488 Phalloidin Conjugate working solution

Add 1 µL of XFD488 Phalloidin 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 XFD488 Phalloidin 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 FITC filter set.

Spectrum

Open in Advanced Spectrum Viewer

Spectral properties

Correction Factor (260 nm)	0.3
Correction Factor (280 nm)	0.11
Extinction coefficient (cm ^-1 M ^-1)	73000
Excitation (nm)	499
Emission (nm)	520
Quantum yield	0.92¹

Product Family

Name	Excitation (nm)	Emission (nm)	Extinction coefficient (cm ^-1 M ^-1)	Quantum yield	Correction Factor (260 nm)	Correction Factor (280 nm)
XFD488 azide Same Structure to Alexa Fluor™ 488 azide	490	520	71000	0.92¹	0.30	0.11
XFD488 alkyne Same Structure to Alexa Fluor™ 488 alkyne	490	520	71000	0.92¹	0.30	0.11
XFD488 Hydroxylamine Same Structure to Alexa Fluor™ 488 Hydroxylamine	490	520	71000	0.92¹	0.30	0.11
XFD488 amine Same Structure to Alexa Fluor™ 488 amine	490	520	71000	0.92¹	0.30	0.11
XFD350 Phalloidin XFD350 Same Structure to Alexa Fluor™ 350	343	441	19000	-	0.25	0.19
XFD594 Phalloidin XFD594 Same Structure to Alexa Fluor™ 594	590	618	92000	0.66¹	0.43	0.56
XFD488 acid Same Structure to Alexa Fluor™ 488 acid	490	520	71000	0.92¹	0.30	0.11
XFD488 tetrazine Same Structure to Alexa Fluor™ 488 tetrazine	490	520	71000	0.92¹	0.30	0.11
XFD488 aldehyde Same Structure to Alexa Fluor™ 488 aldehyde	490	520	71000	0.92¹	0.30	0.11
XFD488 TCO	499	520	73000	0.92¹	0.3	0.11
XFD647 Phalloidin equivalent to Alexa Fluor® 647 phalloidin	604	671	239000	0.33¹	0.00	0.03
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Images

Figure 1. Fixed and stained HeLa cells.
HeLa cells were fixed with 4% formaldehyde, permeabilized, and blocked. F-actin were stained with XFD488 phalloidin (Cat No. 23153) and nuclei labeled with Nuclear Red™ DCS1 (Cat No. 17552). Images were acquired on a Keyence BZ-X710 all-in-one fluorescence microscope.

Citations

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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
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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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Journal: Toxicol Appl Pharmacol (2009): 13

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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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Journal: Cold Spring Harb Protoc (2009): pdb prot5283