7. Phalloidin, green fluorescence, FITC-labeled scorpion toxin (green fluorescence) $ 599

Product Information

Product Name: FITC-labeled Phalloidin (Green Fluorescence) Product Number: G1028-100T Size: 100 tests

Product Description

Phalloidin, also known as Phalloidin toxin, is a bicyclic peptide initially isolated from the toxic mushroom Amanita phalloides. It exhibits a high affinity and specificity for binding to F-actin, a component of muscle fibers, with no binding to G-actin. Phalloidin demonstrates similar affinities for different fiber sizes, and its binding ratio typically involves one phalloidin molecule per actin subunit. Phalloidin has minimal non-specific binding, resulting in distinct staining of both stained and unstained regions. It can stain F-actin at nanomolar concentrations, making it useful for labeling, identifying, and quantifying F-actin distribution.

This product is FITC-labeled Phalloidin provided in a 20 μM storage solution format with DMSO as the solvent. The recommended working concentration range is 80-200 nM, using 200 μL of working solution per staining, resulting in a 100 nM working concentration. This product allows for 100 cell staining procedures.

Storage and Transportation

Shipped with ice packs (wet ice) and stored at -20°C in the dark. Shelf life is 12 months.

Components

• G1028-100T FITC-labeled Phalloidin (Green Fluorescence) 100 tests
• Instruction Manual 1 copy

Preparation Before Experiment

Prepare 1× PBS buffer (pH 7.2-7.4, recommended G4202), BSA (recommended GC305010), fixative solution (containing 4% paraformaldehyde, recommended G1101), permeabilization solution (0.1%-0.5% Triton X-100 in PBS, recommended G1204), anti-fade mounting medium (recommended G1401), and ready-to-use DAPI staining solution (recommended G1012), etc.

For the first use of this product, thaw the product thoroughly and centrifuge at low speed for 1 minute to prevent liquid loss along the tube wall. It is recommended to aliquot based on the amount needed for a single experiment to prevent solvent evaporation loss. Store at -20°C in the dark.

Before the formal experiment, prepare 1% BSA-containing PBS by dissolving 1.0 g of BSA in 100 mL of PBS. Mix 1 μL of FITC-labeled Phalloidin with 199 μL of 1% BSA-containing PBS to obtain a 100 nM working solution. The commonly used working concentration is 80-200 nM, with 100 nM recommended. The optimal staining concentration can be determined through literature references or preliminary experiments. The working solution of FITC-labeled Phalloidin can be used on the same day, stored at room temperature in the dark.

Experimental Procedure

1. Plate cultured cells (density at least half-confluent), remove the culture medium, and wash cells twice with pre-warmed 1× PBS buffer at 37°C.
2. Cover cells with fixative solution containing 4% paraformaldehyde at room temperature and fix for 10-30 minutes. Note that methanol can disrupt actin, so the fixative solution should not contain methanol.
3. Wash cells with 1× PBS buffer at room temperature for 2-3 times, 10 minutes each time.
4. Cover cells with permeabilization solution at room temperature and permeabilize cells for 5 minutes.
5. Wash cells with 1× PBS buffer at room temperature for 2-3 times, 10 minutes each time.
6. Gently shake the cell plate to position cells in the center of the circle. Cover cells completely with 200 μL of the freshly prepared FITC-labeled Phalloidin working solution and incubate at room temperature in the dark for 30 minutes. To prevent solution evaporation and maintain humidity during incubation, place the cell plate in a sealed container, such as a light-protected humidified chamber. Additionally, adding 1% BSA to the Phalloidin staining solution can effectively reduce background staining.
7. Wash cells with 1× PBS buffer for 2-3 times, 5 minutes each time.
8. (Optional) Cover cells with ready-to-use DAPI staining solution for 3-5 minutes to stain cell nuclei. Wash cells with 1× PBS buffer for 2-3 times, 30 seconds to 1 minute each time.
9. Gently dry the cell plate, invert it onto a glass slide with a drop of anti-fade mounting medium, and remove excess mounting medium with a tissue.

[Optional step]: After completing step 7, you can also directly place the cover glass slide with DAPI-containing anti-fade mounting medium (recommended G1407) onto the cell plate, then remove excess mounting medium. Observe the results using a fluorescence microscope or confocal laser scanning microscope.

10. Observe the results using a fluorescence microscope or confocal laser scanning microscope, and select FITC (Ex/Em=492/518 nm) and DAPI (Ex/Em=364/454 nm) channels.

Notes

1. Fluorescent dyes can undergo quenching, so it is advisable to complete detection and observation soon after staining.
2. Methanol can disrupt actin, so fixative solutions containing methanol should not be used during initial fixation.
3. Phalloidin generally lacks cell permeability and is rarely used for live cell staining.
4. The solvent of this product is methanol, which evaporates easily. Seal the cap tightly after each use to prevent evaporation.
5. Wear laboratory attire and disposable gloves when performing the procedure.

This product is for research purposes only and not intended for clinical diagnosis.