Cell Signaling Technilogy
Anti-caspase 8 antibody
9746
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To test the apoptotic, necroptotic, and authopagic protein targets implicated in human normal fibroblasts treated with 3-MA, Western blotting was performed. Briefly, cells were seeded in 2% FBS medium overnight. The cells were treated with 1 mM 3-MA for 4 hours, and the cells were collected and lysed with the buffer [1% SDS, 10 mmol/L Tris-Cl (pH 7.6), 20 μg/μL aprotinin, 20 μg/μL leupeptin, and 1 mmol/L 4-(2-aminoethyl)benzenosulfonyl fluoride]. The protein concentrations were determined using the Bicinchoninic Acid Protein Assay kit (Pierce, Rockford, IL). Fiften micrograms of protein were separated on SDS-PAGE gels and transferred to polyvinylidene difluoride membranes. After blocking, the membranes were incubated with the appropriately diluted primary antibody at 4°C overnight. After washing with TBST, the membranes were incubated with appropriately diluted HRP-conjugated secondary antibodies at room temperature for 1 hour. Proteins were detected with the enhanced chemiluminescence kit (Amersham Pharmacia Biotechnology, Inc., Piscataway, NJ).To screen the proteins complicated in mouse lung ischemia, the lungs from B6 wild type mice were procured and subject to cold ischemia for 24 hours. The lung tissue lysates were made by adding lysis buffer [1% SDS, 10 mmol/L Tris-Cl (pH 7.6), 20 μg/μL aprotinin, 20 μg/μL leupeptin, and 1 mmol/L 4-(2-aminoethyl)benzenosulfonyl fluoride] after homogenizing with mortar and pestle in liquid nitrogen. The supernatants were obtained after centrifugation at 12000 g for 15 minutes at 4. The protein concentrations were determined using the Bicinchoninic Acid Protein Assay kit (Pierce, Rockford, IL). Fiften micrograms of protein were separated on SDS-PAGE gels and transferred to polyvinylidene difluoride membranes. After blocking, the membranes were incubated with the appropriately diluted primary antibody at 4°C overnight. After washing with TBST, the membranes were incubated with appropriately diluted HRP-conjugated secondary antibodies at room temperature for 1 hour. Proteins were detected with the enhanced chemiluminescence kit (Amersham Pharmacia Biotechnology, Inc., Piscataway, NJ).
Western Blot
Human normal fibroblasts
1/1000 dilution 4°C overnight
5% milk
1/1000 diution
Room temperature 1 hour
ECL
A specific band around 50 kDa was detected
N/A
The antibody worked
The antibody worked after optimization