Anti ha peroxidase high affinity antibody

Cell-free translation reactions using WGE were carried out as described previously (41 (link)). For the puromycin reactions in WGE, puromycin was added at a final concentration of 2 mM and then the reaction mixtures were incubated at 25°C for the time-periods indicated for each experiment. For the RNase A treatment, RNase A was added at a final concentration of 0.5 mg/ml and then the reaction mixtures were incubated for 15 min at 37°C.
For the immunoblot analysis, samples were separated on 12% SDS-PAGE. Reaction mixtures were diluted with the 1× SDS-PAGE gel sample buffer (50 mM Tris–HCI pH 6.8, 50 mM DTT, 10% [v/v] glycerol and 1% (w/v) SDS). Translation samples were incubated for 10 min at 67°C and then loaded on a 12% BisTris gel (0.33 M Bis-Tris, 12% acrylamide:bisacrylamide 37.5:1) in MES-SDS running buffer (2.5 mM MES, 2.5 mM Tris Base, 0.005% SDS, 0.5% EDTA, pH 7.1). The samples were subsequently transferred to an Immobilon-PSQ membrane (Merck Millipore, Burlington, MA, USA) and probed using the monoclonal Anti-HA-Peroxidase, High Affinity antibody (Roche, Basel, Switzerland). The immuno-reacted bands were visualized using the Immobilon Forte Western HRP substrate (Merck Millipore) and LuminoGraph I (ATTO, Tokyo, Japan). Digital data were acquired using CS Analyzer 4 (ATTO).