Anti ddddk tag monoclonal antibody

Proteins were separated by SDS-PAGE using 10% or 12% polyacrylamide gel. The gels were then subjected to western blotting. Proteins were transferred onto a polyvinylidene fluoride (PVDF) membrane using the Mini Trans-Blot Electrophoretic Transfer Cell (Bio-Rad, Hercules, CA, USA). The blocking step was carried out in 5% skim milk in Tris-buffered saline containing 0.1% Tween 20 (TBST, pH 7.6), followed by the incubation of the membrane with each primary antibody, namely, mouse anti c-Myc monoclonal antibody (FUJIFILM Wako Pure Chemical, Osaka, Japan), anti-DDDDK-tag monoclonal antibody (Medical & Biological Laboratories, Nagoya, Japan) or rat anti-PA tag monoclonal antibody (FUJIFILM Wako Pure Chemical). Each primary antibody was diluted 1,000-fold before use. After washing with TBS-T, the membrane was incubated with each secondary antibody, namely, 10,000-fold-diluted sheep HRP-linked IgG (GE Healthcare Japan, Tokyo, Japan) or goat anti-rat IgG-HRP (Santa Cruz Biotechnology, Dallas, USA). Detection based on the HRP reaction was carried out using Immobilon Western Chemiluminescent HRP Substrate (Merck Millipore Japan, Tokyo, Japan). Protein bands were detected on a Fluor-S MAX MultiImager (Bio-Rad).