Gene Tex
Anti-caspase 8 antibody
GTX110723
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To examine the expression levels of the apoptotic, necroptotic, and autophagic protein targets implicated in human normal fibroblasts and human multiple myeloma cells, 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). Fifteen 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 and human multiple myeloma cells
1/1000 dilution 4°C overnight
5% milk
1/2000 diution
room temperature 1 hour
ECL
bands of 43 kDa and 57 kDa were detected
N/A
The antibody worked
the antibody worked