Mouse monoclonal anti t7

Purified proteins were added to a continuous sucrose gradient. Continuous sucrose gradients were prepared by overlaying 2.5 ml of 17.5% sucrose in gradient reaction buffer (GRB; 20 mM Hepes, pH 7.0, and 150 mM NaCl) with 2.5 ml of 2.5% sucrose in GRB in a 13 × 51 mm polyallomer centrifuge tube (Beckman Coulter). The centrifuge tube was sealed with parafilm, set horizontally for 2 h at room temperature, then set vertically for 1 h at 4°C. A molecular weight marker mix (HMW Native Marker kit; GE Healthcare) was added to a separate continuous sucrose gradient. Sucrose gradients were centrifuged in an SW55 rotor (Beckman Coulter) at 100,000 g at 4°C for 16 h. Then, 500-µl fractions were pipetted from the top and the pellet was resuspended in 500 µl of 17.5% sucrose in GRB, resulting in a total of 11 fractions. Equal volumes of each fraction from the molecular weight marker gradients were analyzed by SDS-PAGE followed by silver stain analysis. Equal volumes of each fraction from the purified protein gradients were analyzed by SDS-PAGE followed by Western blot analysis using mouse monoclonal anti-T7 (EMD Millipore) for the primary antibody and goat anti–mouse IgG Dylight 680 (Thermo Fisher Scientific) for the secondary antibody. The immunoreactive bands were detected with the Odyssey Infrared Imaging System (LI-COR Biosciences).