Amersham ecl western blot detection kit

P. falciparum 3D7 and FC27 (D10 clone) strains were cultured in human erythrocytes at a hematocrit of 3% in RPMI media supplemented with 10% AlbuMAX II. Infected erythrocytes were harvested at the schizont stage of parasite development at 10% parasitaemia, then lysed with 0.1% saponin. Parasite lysates were fractionated by SDS-PAGE, then transferred to nitrocellulose membranes. Blots were blocked with 5% skim milk, incubated with a 1:500 dilution in 5% skim milk of pooled sera from each group of immunized mice, washed with PBS containing 0.05% Tween-20 (PBS-T), incubated with 1:5,000 goat anti-mouse IgG HRP, washed with PBS-T and developed with Amersham ECL Western Blot detection kit (GE Healthcare).

Western blot (WB) analysis was performed to examine levels of c-Met protein, in which the same number of c-Met-BMSCs and control BMSCs (transfected with lenti-GFP empty vector) were used to extract total protein samples. Proteins were then separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto nitrocellulose membranes as previously reported [6 (link)]. The c-Met protein was detected with the rabbit anti-c-Met (SP260) primary antibody (Santa Cruz Biotechnology, Santa Cruz, USA) with mouse anti-actin antibody as control antibody (Santa Cruz Biotechnology, Santa Cruz, USA) for detection of the internal control protein, beta-actin. The secondary antibodies used in the WB analysis were a horseradish peroxidase (HRP)-conjugated donkey anti-rabbit IgG and a HRP-conjugated ECL sheep anti-mouse IgG (GE Healthcare Biosciences, UK). The resulting blots were detected by enhanced chemiluminescence (ECL) using an Amersham ECL Western blot detection kit (GE Healthcare Biosciences, Pittsburgh, USA) and visualized under an imaging system.