Hemolymph and Tissue Protein Extraction from Oyster

Hemolymph samples from 10 adult C. gigas were prepared as described previously (39 (link)). After centrifugation at 800 g, 4°C for 10 min, supernatant was collected, and hemocytes were pelleted. Hemolymph was centrifuged at 12,000 g, 4°C for 10 min to remove cell debris. Hemocytes were washed with PBS (pH 7.2) for three times at 800 g, 4°C for 10 min, and lysed in RIPA buffer (50 mM Tris–HCl, pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 0.5% deoxycholate and 0.1% SDS) on ice for 15 min. The cell lysate was collected after centrifugation at 12,000 g, 4°C for 10 min. Hepatopancreas, gill, mantle, and adductor muscle from 10 C. gigas were collected and homogenized in RIPA buffer using Dounce tissue grinders (Sigma, USA). The supernatant was collected by centrifugation at 12,000 g, 4°C for 20 min. The protein concentration was determined by BCA Protein Assay Kit (Pierce, USA). The samples with same amount of proteins (30 µg) were separated by SDS-PAGE. The proteins were transferred from the gel to the polyvinylidene difluoride membranes (Millipore, USA), and the membrane was soaked with 5% BSA in TBST. The membrane was then incubated with antibodies against rCgDM9CP-1 and β-tubulin for 1 h, respectively, followed by HRP labeled secondary antibodies incubation for 1 h. The immune-reactive protein bands were visualized by using an enhanced chemiluminescence kit (Pierce, USA).