Western Blot Analysis of Protein Expression

Third instar larvae or S2 cells were lysed with lysis buffer and boiled with sample loading buffer at 95 °C for 10 mins [39 (link)]. Prepared samples were loaded on 7.5% SDS-PAGE gel, and protein bands in the gel were transferred to nitrocellulose membrane by wet method. Membranes were blocked with 5% dry milk in TBS-T (140 mM NaCl; 3 mM KCl; 25 mM Tris pH 7.4; 0.1% Tween-20) at room temperature (RT) for 1 h. Membranes were sequentially probed in primary antibody and HRP-conjugated secondary antibody solution diluted with 2% dry milk in TBS-T. After washing, membranes were developed with WESTSAVE-Gold reagent (AbFrontier).
Primary antibodies used are: concentrated anti-Wg (mouse, 1:2000, Developmental Studies Hybridoma Bank (DSHB)), anti-GFP (rabbit, 1:10,000, abcam), anti-αTub (mouse, 1:5000, Sigma-Aldrich), anti-MYC (rabbit, 1:5000, abcam) and anti-Ago (guinea pig, 1:5000, gift from K. H. Moberg). Secondary antibodies used are: HRP-conjugated anti-mouse antibody (1:10,000, Jackson Laboratory), HRP-conjugated anti-rabbit antibody (1:10,000, Jackson Laboratory) and HRP-conjugated anti-guinea pig antibody (1:5000, Jackson Laboratory).