Anti ras antibody

Following the treatment of the AsPC-1 cells for 48 h, the control and treated cells were collected and lysed with RIPA buffer (Merck) and Complete Protease Inhibitor (Sigma) on ice for 20 min. Protein fractions were separated by centrifugation for 10 min at 13,000 rpm at 4°C. The proteins, in 20-µg aliquots, were separated by SDS-PAGE and then transferred by semi-dry transfer to 0.45-micrometer-pore-size nitrocellulose membranes. The membranes were blocked with 5% milk TBST solution for 1 h at room temperature, followed by overnight incubation with rabbit monoclonal Anti-Ras Antibody (Abcam). They were then washed three times with TBST, each for 10 min, incubated with secondary antibody, peroxidase-conjugated, AffiniPure Goat Anti-Rabbit IgG (Jackson Immune Research Laboratories and Dako) for 1 h at room temperature, and washed three more times. An antigen/antibody complex was detected with an ECL kit Western Bright^™ ECL detection reagent (Advansta). The results were analyzed with an Amersham Imager 600 (GE Healthcare).
For reference, and after detection with the specific antibodies, all of the membranes were exposed to mouse Anti-β-actin monoclonal antibody (MP Biomedicals). Donkey polyclonal anti-mouse HRP secondary antibody and an ECL kit were used to detect actin.