Caspase-12Data Courtesy of Ramaswamy Sharma and Dr. Alexander Gow, Wayne State University. |
Samples were loaded onto a 12% separating gel. After transfer, blots were blocked in 5% milk for 1 hour; primary antibody (rabbit anti-Caspase-12, 1:3000, RA15048) was added in 3% milk and the antibody was allowed to bind overnight at 4C. After washing 3 times in 1XPBST (10 mins each), a secondary antibody (goat anti-rabbit IgG-biotin, 1:2000, Southern Biotechnology Associates, Inc.) was added for 1 hour at RT. Blot was washed again in 1X PBST for 3 times (10 mins each), followed by the addition of tertiary antibody (SAV-Alkaline Phosphatase, 1:2000, Jackson Immunoresearch Labs, Inc.) in 3% milk for 1 hour at RT. After washing 3 times in 1X PBST (10 mins each), the blot was rinsed in 100 mM Tris, pH 9.5 and substrate (NBT/BCIP, Roche) was added. The blot was stripped and reprobed for b-actin (Sigma, cat# A1978). Dr. Yuan kindly provided the Caspase-12 null mice.
Background Animal References:
Nakagawa T, Zhu H, Morishima N, Li E, Xu J, Yankner BA, Yuan J. Caspase-12 mediates endoplasmic-reticulum-specific apoptosis and cytotoxicity by amyloid-b. Nature 2000 January 6;403:98-103
Southwood CM, Garbern J, Jiang W, Gow A (2002). The unfolded protein response modulates disease severity in Pelizaeus-Merzbacher disease. Neuron 36:585-96.
