2 de clean up 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).

Acetonitrile, trifluoroacetic acid (TFA), K₃Fe(CN)₆, HCCA, 5-hydroxydecanote (5-HD), Nycodenz and urea were purchased from Sigma (St. Louis, MO, USA). Bromophenol blue, acrylamide, CHAPS, immobilized pH gradient (IPG) strips, two-dimensional polyacrylamide gel electrophoresis (2DE) Quant kit, 2DE Clean-up Kit, polyvinylidene fluoride membrane and low melting point agarose were purchased from Bio-Rad (Hercules, CA, USA). Glycerol, glycine, ammonium persulfate, N, N′-methylene acrylamide, SDS, Tris, EDTA, sucrose and mannitol were supplied by Amresco (Solon, OH, USA). Secondary antibodies and anti-GRP75, anti-NDUFV1, anti-DLD and anti-cytochrome oxidase IV (COX IV) primary antibodies were obtained from Abcam (Cambridge, UK). TEMED was from Amersham (UK). Butanol was from Merck (Darmstadt, Germany). Trypsin was from Promega (Madison, WI, USA). Pentobarbital sodium and Tween 20 were both from Solarbio (Beijing, China). All other unlisted reagents were of analytical grade or guaranteed reagents.