Acrylodan, when subjected to thiol reactions, exhibits sensitivity to the dipolarity and dynamics of its surrounding binding pocket, specifically those surrounding Cys 34.

Guidelines (Following is our recommended protocol. This protocol only provides a guideline, and should be modified according to your specific needs). Purification and Labeling of Act1: 1. 20-fold molar excess of acrylodan (Anaspec) was added to the polymerized actin and incubated on ice overnight. 2. The labeled actin is centrifuged (2 h, 4 °C, 100,000 × g) then the pellet was resuspended in 10 mm Tris, pH 7.5, 0.5 mm β-mercaptoethanol, 0.2 mm CaCl2, 50 μm ATP and dialyzed against the same buffer for 70 h at 4 °C. 3. The supernatant was collected and the actin concentration and degree of labeling were calculated using the extinction coefficient 26,600 M ?1 cm ?1 for actin at 290 nm, 18,500 M ?1 cm ?1 or acrylodan at 385 nm, and 71,000 M ?1 cm ?1 for Alexa Fluor 488 at 494 nm. 4. Acrylodan-labeled Act1 ( Acryl Act1 NAT ) is dramatically different than that of EDTA-unfolded Acryl Act1 ( Acryl Act1I3), which has a very similar emission spectrum to CCT-bound Acryl Act1.