Goat anti mouse secondary antibody

A lung cancer tissue microarray (TMA) was purchased from Chaoying Biotechnology Co. (Xi’an, China). Sections were incubated with anti-IQGAP3 antibody (1∶50 dilution) overnight at 4°C. The primary antibody was identified using a horseradish peroxidase enzyme-labeled polymer conjugated to goat anti-mouse secondary antibody (Promega). The stained sections were reviewed and scored by a pathologist. The negative control was normal human lung tissue.

Protein samples were resolved by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS–PAGE), and transferred to a PVDF membrane. Membranes were probed using purchased mouse monoclonal anti-His-tag antibody (1:1,000; Roche, USA). Detection of the protein was carried out using horseradish peroxidase conjugated to goat anti-rabbit or goat antimouse secondary antibody (1:5,000; Promega) with the DAB Horseradish Peroxidase Color Development Kit (Beyotime, China).

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed on 10 or 4–12% gradient acrylamide gels. Generally, gels were ran for 1 h at 160 V. Total proteins were visualized by Coomassie brilliant blue staining for 1 h and destained until the background became transparent.
For immunoblotting, after SDS-PAGE, proteins were transferred onto polyvinylidene fluoride membrane (Millipore) for 2 h 30 min at 300 mA. Membranes were blocked in 5% non-fat milk (dissolved in TBST buffer containing 25 mM Tris, 250 mM NaCl, 0.1% Tween-20, pH 7.4) for at least 2 h at room temperature. For detection of soluble gp350-ECD₁₂₃-6His, primary mouse anti-His antibody (Abmart) was diluted 1:2,000 and incubated with membrane at 4°C overnight following incubation with secondary goat anti-mouse antibody (1:20,000, Promega) conjugated to horseradish peroxidase (HRP) for 1 h at room temperature. For detection of Fc fusion proteins, the membranes were incubated directly with secondary goat anti-mouse antibody (1:20,000) for detection using ECL substrate (Advansta).