Cellular Dyes
Alexa Fluor® 555 Phalloidin #8953
Directions for Use
To make a stock concentration of 6.6 μM (20X), reconstitute the lyophilized material in 1.5 ml pure methanol.
Fix cells for 15 minutes using fresh, methanol-free 4% formaldehyde, then rinse three times in PBS for 5 minutes each. Once fixed, please follow CST protocol for immunostaining. Following incubation of the primary and secondary antibodies, Alexa Fluor® 555 Phalloidin can be diluted 1:20 (5 μl per 100 μl assay volume) in PBS and added to the cells. Allow to incubate for 15 minutes at room temperature, then rinse once with PBS. Coverslip slides with ProLong® Gold antifade reagent and examine specimen using appropriate excitation wavelength.
IF-IC
Confocal immunofluorescent analysis of HeLa cells using COX IV (3E11) Rabbit mAb (Alexa Fluor® 488 Conjugate) #4853 (green). Actin filaments were labeled with Alexa Fluor® 555 Phalloidin (red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).
Description
Alexa Fluor® 555 Phalloidin allows researchers to fluorescently stain the cytoskeleton through the binding of phalloidin to F-actin. This product is intended for use on fixed and permeabilized samples due to the toxicity associated with phalloidin. After reconstitution the stock solution provides enough material to perform 300 assays based on a 1:20 dilution and a 100 μl assay volume.Alexa Fluor® 555 Fluorescent Properties: Excitation: 555, Emission: 565.
Background
Actin, a ubiquitous eukaryotic protein, is the major component of the cytoskeleton. At least six isoforms are known in mammals. Nonmuscle β- and γ-actin, also known as cytoplasmic actin, are predominantly expressed in nonmuscle cells, controlling cell structure and motility (1). Actin exists mainly as a fibrous polymer, F-actin. In response to cytoskeletal reorganizing signals during processes such as cytokinesis, endocytosis, or stress, cofilin promotes fragmentation and depolymerization of F-actin, resulting in an increase in the monomeric globular form, G-actin (2). Phalloidin is a natually occuring toxic bicyclic peptide found in the deathcap toadstool, Amanita phalloides, that rapidly binds to F-actin with strong affinity (3).
- Herman, I.M. (1993) Curr Opin Cell Biol 5, 48-55.
- Condeelis, J. (2001) Trends Cell Biol 11, 288-93.
- Lengsfeld, A.M. et al. (1974) Proc Natl Acad Sci U S A 71, 2803-7.
Application References
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Companion Products
The transfer of this product is contingent on the buyer using the purchased product solely in research conducted by the buyer (whether the buyer is an academic or for-profit entity), for immunocytochemistry, high content screening (HCS) analysis, or flow cytometry applications. The sale of this product is expressly conditioned on the buyer not using the product or its components (1) in manufacturing; (2) to provide a service, information, or data to an unaffiliated third party for payment; (3) for therapeutic, diagnostic or prophylactic purposes; (4) resale, whether or not such product or its components are resold for use in research; or for any other commercial purpose. For information on purchasing a license to this product for purposes other than research, contact Life Technologies Corporation, 5791 Van Allen Way, Carlsbad, CA 92008 USA or outlicensing@lifetech.com. Alexa Fluor® is a registered trademark of Molecular Probes, Inc.
For Research Use Only. Not For Use In Diagnostic Procedures.
