A Microplate-Based Nonradioactive Protein Synthesis Assay: Application to TRAIL Sensitization by Protein Synthesis Inhibitors.

Curtis J Henrich

PloS one 2016

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Non-radioactive assays based on incorporation of puromycin into newly synthesized proteins and subsequent detection using anti-puromycin anti bodies 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.

Citation

Curtis J Henrich. A Microplate-Based Nonradioactive Protein Synthesis Assay: Application to TRAIL Sensitization by Protein Synthesis Inhibitors. PloS one. 2016;11(10):e0165192