Acrylodan

Acrylodan, reacted with thiols, is sensitive to the local environmental dipolarity and dynamics within the binding pocket surrounding Cys^34[1][2]. In Vitro: 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 (^AcrylAct1_NAT) is dramatically different than that of EDTA-unfolded ^AcrylAct1 (^AcrylAct1I3), which has a very similar emission spectrum to CCT-bound ^AcrylAct1.