Hrp conjugated secondary antibody

Cells were harvested and lysed with RIPA buffer (Cell signaling, 9806) in ice for 30 min and centrifuged at 12000× g for 10 min. Supernatant was collected and protein concentration was measured using BCA method (Thermo Fisher Scientific). Protein samples were boiled in 1x NuPAGE LDS sample buffer (Invitrogen) at 70℃ for 10 min. Twenty microgram protein samples were subjected to 7.5% - 15% gradient SDS-PAGE gel and transferred to PVDF membrane (MACHEREY-NAGEL, Germany). The membranes were blocked in 1x Roti-Block (Carlroth, Germany) at room temperature for 1 h and were then probed with specific primary antibodies overnight at 4°C. Blots were incubated with HRP-conjugated secondary antibody (Invitrogen, 31430 and 31460) for 1 h at room temperature. Bands were visualized by SuperSignal West Pico PLUS Chemiluminescent Substrate (Thermo Fisher Scientific, USA) and detected using the ChemoStar ECL Imager (Intas Science Imaging, Germany).

Following the indicated treatments, cells were washed once in ice-cold PBS and proteins were collected in lysis buffer (20 mM Tris-HCl pH 8.0, 1% NP-40, 150 mM NaCl, 2 mM EDTA) supplemented with protease inhibitor cocktail (Roche Diagnostics Scandinavia AB, Bromma, Sweden) and cleared by centrifugation at 14,000x g at 4 °C for 10 min. Lysate protein concentration was measured by a Bradford assay. Equal protein amounts from each sample were separated with sodium dodecyl sulphate-polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane. Membranes were blocked for 1 h in 5% milk/TBS-T and incubated overnight at 4 °C with primary antibody in TBS-T. Following 3 washes in TBS-T, corresponding HRP-conjugated secondary antibody (Invitrogen/Life Technologies Corp., Foster City, CA, USA) was added in TBS-T at a concentration of 1:20,000 (anti-mouse) or 1:40,000 (anti-rabbit) and incubation prolonged for 1 h at room temperature. Antibody binding was visualised with the enhanced chemiluminescence detection system (Thermo Fischer Scientific Inc., Waltham, MA, USA). Images were captured with a Fuji scanner using the AIDA software (Fuji Inc.) and band intensities were calculated using Photoshop CS3.