Review for Anti-Cereblon Antibody (clone 3B12F7)

The rough idea is based on the literature. Accumulating evidence showed that the imid (thalidomide, lenalidomide, and pomalidomide) is targeting cereblon (crbn). And ikaros (ikzf1) is one of the substrate of crbn. We are in the process to validate the quality of the crbn antibody, and its target ikaros. We are trying to test if ido1 is one of the substrate of crbn. Cells were 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). Ten micrograms of protein were separated on SDS-PAGE gels and transferred to polyvinylidene difluoride membranes. After blocking, the membranes were incubated with the appropriate primary antibody at 4°C overnight. After washing with TBST, the membranes were incubated with HRP-conjugated secondary antibodies at room temperature for 1 hour. Proteins were detected with the enhanced chemiluminescence kit (Amersham Pharmacia Biotechnology, Inc., Piscataway, NJ).