Applying the ribopuromycylation method to detect nuclear translation

Abstract

Protein translation in the nucleus has been controversial for more than four decades. To take a new look at this potentially important phenomenon, we adapted the RiboPuromycylation Method (RPM) which labels actively translating ribosomes in cells via standard immunofl uorescence microscopy. RPM is based on puromycylation of nascent chains trapped on ribosomes by antibiotics which inhibit chain elongation, followed by cell permeabilization/fixation and detection of puromycylated nascent chains using a puromycin- specific monoclonal antibody. To adapt the method to the nucleus, we use NP-40 rather than digitonin to permeabilize cells because NP-40 enables better antibody penetration into the nucleoplasm and particularly the nucleoli, a region of high translation as shown by RPM.