Electrochemiluminescence detection kit

FLSs were incubated with IL-17. After incubation for 72 hours, whole-cell lysates were prepared from approximately 2 Â 10 5 cells by homogenization in the lysis buffer, and centrifuged at 19,320 Â g for 15 minutes. The protein concentration in the supernatant was determined using the Bradford method (Bio-Rad, Hercules, CA). Protein samples were separated onto 10% SDS-PAGE, and transferred to a nitrocellulose membrane (Amersham Pharmacia Biotech, Uppsala, Sweden). For western hybridization, the membrane was preincubated with 0.5% skim milk in 0.1% Tween 20 in Tris-buffered saline at room temperature for 2 hours. The primary antibody to RANKL (R&D Systems), diluted 1:1000 in 5% bovine serum albumine0.1% Tween 20/Tris-buffered saline, was added and incubated for overnight at 4 C. The membrane was washed four times with 0.1% Tween 20 in Tris-buffered saline, and horseradish peroxidaseeconjugated secondary antibody was added and incubated for 1 hour at room temperature. After 0.1% Tween 20 in Tris-buffered saline washing, hybridized bands were detected using the electrochemiluminescence detection kit and Hyperfilm-enhanced chemiluminescence (ECL) reagents (Amersham Pharmacia Biotech, Piscataway, NJ).