Pierce 660 kit

Individuals of A. vulgaris were frozen immediately after harvest in liquid nitrogen and stored at -80°. Mantles were dissected directly before protein extraction procedure and homogenized in urea buffer (7 M urea, 2 M thiourea, 4% CHAPS, 2 M Tris (pH 8.8), 0.5% Carrier (3–11 pH), 1% DTT, 1 mM EDTA, 1 mM PMSF, Protease Inhibitor C). Protein samples were cleaned using a 2-DE clean up kit (BioRad, Hercules, CA, USA) and protein concentration was estimated by the Pierce 660 kit (ThermoScientific, Rockford, USA).

The enzymatic parameters of substrate hydrolysis by generic hABHD6 enzyme were evaluated in three different biochemical assays: hydrolysis of 2-AG to AA and glycerol was quantified by either HPLC (1) or a glycerol-based colorimetric method (2), and a fluorometric method using the fluorogenic substrate AHMMCE (3). Prior enzyme assays a total protein concentration in supernatant of solubilized membrane fraction containing of full-length hABHD6 was determined using the Pierce 660 Kit (Thermo Fisher Scientific, Pittsburgh, PA). The assays to measure protein concentrations, intensity of fluorescence or color of formed products were performed in a 96-well plate format using a Synergy HT (BioTek Instruments, Winooski, VT) or EnVision (Perkin Elmer, Waltham, MA) plate readers. All activity assays were performed in triplicate and the Michaelis-Menten kinetic parameters were calculated using Prism software, Version 5 (Graphpad, San Diego, CA).