Dimethyl pimelimidate dihydrochloride dmp

Ten milliliters of hybridoma supernatant was added to 50 μl of slurry Protein G Sepharose beads and rotated at room temperature for 1 h. The Protein G Sepharose-antibody beads were washed with borate buffered saline (0.2 M boric acid/NaOH pH 9.0) three times and rotated with 5.2 mg/ml dimethyl-pimelimidate-dihydrochloride (DMP, Sigma-Aldrich) in Borate buffered saline for 30 min. The beads were then washed two times with 0.2 M ethanolamine, rotated for 2 h with 0.2 M ethanolamine (pH 8) and washed three times with PBS. Hemocytes were isolated from the hemolymph, by centrifugation (4°C, 1800 rpm, 8 min). The cells were extracted in lysis buffer containing 50 mM Tris/HCl (Reanal) pH 8.0, 150 mM MgCl 2 (Reanal), 1% NP40 (Fluka), 5 mM EDTA (Sigma), 0.1% SDS (Sigma), 10 mM PMSF (Sigma) and Complete Protease Inhibitor Cocktail (Roche), according to the manufacturer's instructions, for one hour. Fifty µl (20% Protein G Sepharose) beads were mixed with 300µl hemocyte lysate and incubated overnight at 4°C. Then the beads were washed three times with lysis buffer and incubated in SDS-PAGE sample buffer (15.6 mM Tris/HCl pH 6.8, 6.25% glycerol, 0.5% SDS, 0.003% bromophenol blue) for one hour at 56°C and subsequently boiled for 5 min. The resulting samples were analyzed with SDS-PAGE/Western blot.