Irdye 680cw goat anti mouse igg secondary antibody

Snap-frozen tumors were digested with radioimmunoprecipitation assay lysis buffer containing phosphatase inhibitors (Sigma). Protein concentrations were measured with a Pierce BCA protein assay (23227, Thermo Scientific, MA, USA). 50 μg of protein per sample was separated on 12% SDS-PAGE and then electro-blotted onto nitrocellulose membranes using the iBlot Dry Blotting System (Thermo Scientific). Membranes were blocked with 3% bovine serum albumin in PBS with 0.02% Tween 20 (PBST) for 1 h and then probed with rabbit anti-survivin antibody (1:10,000 dilution; Ab 76424, Abcam) at 4°C overnight (16 h). Following three washes with PBST, the membrane was incubated with IRDye 800CW goat anti-rabbit immunoglobulin G (IgG) (1:5,000 dilution, 925-32211, LI-COR Biosciences, NE, USA) for 1 h. Expression of GAPDH was used as a loading control using a mouse anti-GAPDH antibody (1:10,000 dilution, MAB374, Sigma) and IRDye 680CW goat anti-mouse IgG secondary antibody (1:5,000 dilution, 926-32220, LI-COR Biosciences). The fluorescent signal was measured using the Odyssey imaging system (LI-COR Biosciences). The same protocol was used to measure survivin levels in cell lysates.