Phalloidin-AF488,IC-6138

Description

This green fluorescent phalloidin conjugate (equivalent to FITC 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. BioCytoSci Inc. offers a variety of fluorescent phalloidin derivatives with different colors for multicolor imaging applications.
Size

300Tests

Form

This product is lyophilized powder, and it is difficult to observe the trace amount. Please centrifuge before use, and add appropriate solvent to dissolve it. The solution after dissolution is almost transparent.

Storage/Stability

Shipped at 4¡æ. Store at -20¡æ and protect from light for 12 months.

Sample Types

Formaldehyde-fixed, permeabilized tissue sections, cell cultures or cellfree experiments.

Usage  Protocol

Staining fixed cells The following protocol describes the staining procedure for adherent cells grown on glass coverslips or 8-well chamber slides. Phalloidins also can be used to stain fixed frozen or paraffin tissue sections, as well as yeast and fungi.

1.Wash cells 3 times with PBS.

2.Fix cells on ice with 4% formaldehyde solution in PBS for 15 minutes. Note: Methanol can disrupt actin during the fixation process. Therefore, it is best to avoid any methanol containing fixatives or other solvent-based fixatives. The preferred fixative is methanol-free formaldehyde.

3.Wash cells 3 times with PBS.

4.Permeabilize cells with 0.5% Triton X-100 in PBS at room temperature for 10 minutes.

5.Wash cells 3 times with PBS.

6.Dilute 1-5 ¦Ìl fluorescent phalloidin stock solution in 200 ¦Ìl PBS for each cover slip or chamber to be stained. Place the staining solution on the coverslip for 20 minutes at room temperature. Note£ºStaining volume can be adjusted according to the sample. To avoid evaporation, keep the coverslips inside a covered container and the chamber slides covered during the incubation.

7.Wash 2-3 times with PBS.

8.Image using fluorescence microscopy. Fluorescent phalloidins are photostable enough to image in PBS, but for best results we recommend mounting with antifade mounting medium. Staining living cells Fluorescently-labeled phalloidin is not cell-permeant and has therefore has not been used extensively with living cells. However, living cells have been labeled by pinocytosis or unknown mechanism. In general, a larger amount of stain will be needed for staining living cells. Alternatively, fluorescent phalloidins have also been injected into cells for monitoring actin distribution and cell motility.