A non‐radioactive high‐throughput assay for the detection of cellular protein synthesis

Abstract

Protein synthesis is an important feature of cell vitality and many drugs are targeting this process. In addition, several targeted tumor drugs, e.g. immunotoxins, contain protein toxins inhibiting protein synthesis. Up to date, no non‐radioactive assay exists for the direct detection of protein synthesis inhibition in cells. Here, puromycin was used to label naive proteins synthesized in cells in 96‐well plates. Puromycin is transferred onto the growing amino acid chain and terminates the protein synthesis. Subsequent fixation and permeabilization allowed for the immunological detection of puromycinylated proteins by an anti‐puromycin antibody. Analyses with several protein synthesis inhibitors as well as targeted toxins revealed a concentration‐dependent inhibition of cellular protein synthesis on four different cells lines. The kinetics of inhibition by the targeted toxins was studied to identify suitable time points for complete protein synthesis inhibition. The results demonstrate the successful development of a non‐radioactive and sensitive assay for the direct detection of protein synthesis inhibition in a 96‐well format. This assay should help to perform more immediate analyses of drugs targeted on protein synthesis and help to replace the commonly used radioactive leucine incorporation assay.