title: A Microplate-Based Nonradioactive Protein Synthesis Assay: Application to TRAIL Sensitization by Protein Synthesis Inhibitors.
abstract: Non-radioactive assays based on incorporation of puromycin into newly synthesized proteins and subsequent detection using anti-puromycin antibodies have been previously reported and well-validated. To develop a moderate- to high-throughput assay, an adaptation is here described wherein cells are puromycin-labeled followed by simultaneously probing puromycin-labeled proteins and a reference protein in situ. Detection using a pair of near IR-labeled secondary antibodies (InCell western, ICW format) allows quantitative analysis of protein synthesis in 384-well plates. After optimization, ICW results were compared to western blot analysis using cycloheximide as a model protein synthesis inhibitor and showed comparable results. The method was then applied to several protein synthesis inhibitors and revealed good correlation between potency as protein synthesis inhibitors to their ability to sensitize TRAIL-resistant renal carcinoma cells to TRAIL-induced apoptosis.
authors: Molecular Targets Laboratory, Center for Cancer Research, National Cancer Institute, Frederick, Maryland, 21702, United States of America.|Basic Science Program, Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research, Frederick, Maryland, 21702, United States of America.
authors: Curtis J
authors: CJ
authors: Henrich
mesh_terms: D045744:Cell Line, Tumor; D006801:Humans; D014176:Protein Biosynthesis / Q000187:drug effects*; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology*; D053221:TNF-Related Apoptosis-Inducing Ligand / Q000378:metabolism*
chemical_list: D011500:Protein Synthesis Inhibitors; D053221:TNF-Related Apoptosis-Inducing Ligand