Mini protean 3 gel electrophoresis system

Samples were diluted in 4× Laemmli sample buffer (Bio-Rad) (277.8 mM Tris-HCI, pH 6.8, 4.4% LDS, 44.4% (v/v) glycerol, 0.02% bromophenol blue), 5% β-mercaptoethanol (Sigma) was added prior to heating at 95°C for 5 min and loaded on 10% SDS gels. Gel electrophoresis was performed using the Bio-Rad Mini-PROTEAN 3 gel electrophoresis system (Bio-Rad Laboratories). Proteins were transferred to nitrocellulose membranes (Amersham Protran) and blocked with 5% fat-free milk powder in 1× PBS, 0.05% Tween-20 (Sigma) for 1 h at RT. Blots were incubated with primary antibodies overnight (Table 1). Blots were then washed in PBS-T and incubated with secondary antibody (anti-mouse HRP) at RT for 2 h. Membranes were washed three times with PBS-T, prior to application of ECL (Biological Industries). Chemiluminescent signal was detected with Chemi-Doc (Bio-Rad). All membranes were probed for β-tubulin as loading control.

Cells were harvested for SDS-PAGE and immunoblotting by scraping into SDS-PAGE sample buffer containing 2% SDS, 100 mM dithiothreitol, 10% glycerol, 0.1% bromophenol blue and protease inhibitors (Complete Roche) in 50 mM Tris–HCl pH 6.8 and heating to 100 °C for 5 min. Other samples were prepared by addition of SDS-PAGE sample buffer and heating to 100 °C for 5 min. Samples were separated on 8–15% (w/v) acrylamide gels and transferred to Protran nitrocellulose membranes (Schleicher and Schuell) using a Mini-PROTEAN 3 gel electrophoresis system and Transblot system (BioRad). The immunoblots were then blocked by incubation in 5% (w/v) dried milk/0.1% (w/v) Tween-20 in Tris buffered saline (TBS) pH 7.5 for 1 h and then probed with primary antibodies diluted in blocking solution for 16 h at 4 °C. Following washing in blocking solution, they were then incubated with horseradish peroxidase-conjugated goat anti-mouse, anti-rabbit or anti-rat Igs (GE Healthcare). Immunoblots were developed using an enhanced chemiluminescence Luminata Forte Western HRP substrate system according to the manufacturer’s instructions (Millipore). Signals on immunoblots were quantified using ImageJ after scanning with an Epson Precision V700 Photo scanner essentially as described by us in previous studies [78 (link)].