Protocol for Taxol Janelia Fluor® 646

Introduction

Taxol (often referred to as Paclitaxel) is a diterpenoid that binds to tubulin. It promotes and stabilizes tubulin polymerization, which accounts for its anti-mitotic and cytotoxic action. The conjugation of a fluorescent dye to taxol offers a convenient way to image the microtubule cytoskeleton of a cell; well known fluorescent probes based on this principle are Flutax 1 (Catalog # 2226) and Flutax 2 (Catalog # 6254).

Taxol Janelia Fluor^® 646 is a tubulin fluorescent probe that incorporates the red fluorogenic dye, Janelia Fluor^® 646 (Catalog # 6148). The fluorogenicity of the dye renders this probe non-fluorescent until bound to tubulin, enabling no-wash experimental protocols. Guidelines for using this product in a no-wash imaging protocol are provided below.

Protocol

• Prepare a stock solution of Taxol Janelia Fluor^® 646 in DMSO. Stock solutions can be aliquoted and stored for up to 1 month at  ≤-20°C.
• Dilute the stock solution into warm media (37°C) and place into a pre-warmed box (for storage in the incubator). Aqueous working solutions should be prepared and used on the same day.
• Apply to live cells at a concentration of 3 μM. Lower concentrations can be used and the concentration should be optimized for individual experiments.
• Incubate for 1 hour at 37°C prior to imaging. If preferred, a washing protocol can be used. Should this step be required, rinse the cells three times with 1x PBS and 2% BSA and apply fresh media prior to imaging.
• Image cells using appropriate filters for Taxol Janelia Fluor^® 646. Excitation maximum = 655 nm; emission maximum = 671 nm.