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XFD555 Phalloidin *equivalent to Alexa Fluor® 555 phalloidin*
Product key features
- Specific F-Actin Binding: Retains high affinity and specificity for filamentous actin in diverse biological systems.
- Bright and Stable Fluorescence: XFD555 offers exceptional signal intensity and photostability for extended imaging.
- Low Background Signal: Minimal nonspecific binding ensures high-contrast actin visualization.
- Versatile Applications: Reliable in fixed tissues, cultured cells, and actin polymerization assays.
- Multiplex Imaging Ready: Compatible with various fluorescent tags and detection systems for co-labeling experiments.
Product description
XFD555 phalloidin is a high-affinity F-actin probe conjugated to our bright, photostable, red-orange fluorescent XFD555 dye. XFD555, which is manufactured by AAT Bioquest, is structurally equivalent to Alexa Fluor™ 555 (ThermoFisher).
Phalloidin, a bicyclic peptide toxin derived from Amanita phalloides (commonly known as the death cap mushroom), is widely recognized for its high-affinity and selective binding to filamentous actin (F-actin). Fluorescently conjugated phalloidin exhibits exceptional specificity for F-actin across a diverse range of species, including both plant and animal systems, with minimal nonspecific interactions. The conjugation of phalloidin to XFD555, a highly photostable and intensely fluorescent red-orange dye, offers an optimal combination of target specificity and fluorescence performance. This conjugate enables high-contrast, low-background imaging, facilitating precise visualization and quantification of F-actin structures in various biological applications.
XFD555 phalloidin can be used to visualize and quantitate F-actin in tissue sections, cell cultures, or cell-free preparations. XFD555 phalloidin staining is fully compatible with other fluorescent stains used in cellular analyses including fluorescent proteins, Qdots, and other iFluor® conjugates including secondary antibodies.
Example protocol
AT A GLANCE
Prepare samples in microplate wells
Remove liquid from samples in the plate
Add XFD555 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 Cy3 filter
Warm the vial to room temperature and centrifuge briefly before opening.
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
Add 1 µL of XFD555 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
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 XFD555 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
Product family
| Name | Excitation (nm) | Emission (nm) | Extinction coefficient (cm -1 M -1) | Quantum yield | Correction Factor (260 nm) | Correction Factor (280 nm) |
| XFD350 Phalloidin *XFD350 Same Structure to Alexa Fluor™ 350* | 343 | 441 | 19000 | - | 0.25 | 0.19 |
| XFD488 Phalloidin *XFD488 Same Structure to Alexa Fluor™ 488* | 499 | 520 | 73000 | 0.921 | 0.3 | 0.11 |
| XFD594 Phalloidin *XFD594 Same Structure to Alexa Fluor™ 594* | 590 | 618 | 92000 | 0.661 | 0.43 | 0.56 |
| XFD555 amine | 553 | 568 | 155000 | 0.11 | 0.08 | 0.08 |
| XFD555 acid | 553 | 568 | 155000 | 0.11 | 0.08 | 0.08 |
| XFD647 Phalloidin *equivalent to Alexa Fluor® 647 phalloidin* | 650 | 671 | 239000 | 0.331 | 0.00 | 0.03 |
| XFD750 Phalloidin *equivalent to Alexa Fluor® 750 phalloidin* | 752 | 776 | 240000 | 0.121 | 0.00 | 0.04 |
References
Authors: Roda, Vinicius Moraes de Paiva and da Silva, Rafael André and Siqueira, Paula Veloso and Lustoza-Costa, Gabriela Jesus and Moraes, Gabriélla Malheiros and Matsuda, Monique and Hamassaki, Dânia Emi and Santos, Marinilce Fagundes
Journal: Experimental eye research (2024): 109745
Authors: Liehr, Thomas and Kankel, Stefanie and Buhl, Eva Miriam and Schröder-Lange, Sarah K and Weiskirchen, Ralf
Journal: Cells (2024)
Authors: Badlowski, Gina A and Boyle, Kelly S
Journal: The Journal of experimental biology (2024)
Authors: Choroszy, Marcin and Środa-Pomianek, Kamila and Wawrzyńska, Magdalena and Chmielarz, Mateusz and Bożemska, Edyta and Sobieszczańska, Beata
Journal: Vascular health and risk management (2023): 399-409
Authors: Schröder, Sarah K and Tag, Carmen G and Weiskirchen, Sabine and Weiskirchen, Ralf
Journal: Methods in molecular biology (Clifton, N.J.) (2023): 55-66
Safety data sheet