Acrylodan, reacted with thiols, is sensitive to the local environmental dipolarity and dynamics within the binding pocket surrounding Cys34[1][2].
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 (AcrylAct1NAT) is dramatically different than that of EDTA-unfolded AcrylAct1 (AcrylAct1I3), which has a very similar emission spectrum to CCT-bound AcrylAct1.
[1]. Sarah F Stuart, et al. A two-step mechanism for the folding of actin by the yeast cytosolic chaperonin. J Biol Chem. 2011 Jan 7;286(1):178-84. [2]. K Flora, et al. Unfolding of acrylodan-labeled human serum albumin probed by steady-state and time-resolved fluorescence methods. Biophys J. 1998 Aug;75(2):1084-96.