Detected Molecule [Ref.] Detection Method Separation Technique Sensitivity/ Quantification Advantages and Disadvantages Methods employing radioactively labeled amino acids [14C]-peptide/ N-acetyl-[14C]-leucine [16,17] Ninhydrin staining/ Scintillation Paper electrophoresis High/Yes (+) low amount of substrate (-) requires synthetic steps to prepare substrate or uses  acetylated aminoacyl-tRNA [14C]-peptide/ [14C]-diacetyl-lysine [18,19] Scintillation Centrifugation High/Yes (+) low amount of substrate (-) requires synthetic steps to prepare substrate or uses acetylated aminoacyl-tRNA Methods employing electrophoretic separation of tRNA tRNA [22,23] [32P]-ATP (Northern blot) PAGE High/Yes (+) allows use of specific and heterogeneous substrates (-) time consuming (-) requires short peptide length tRNA [24,25] Methylene blue Silver staining PAGE Moderate/Yes High/Yes (+) allows use of specific and heterogeneous substrates (+) fast (-) sensitivity (-) requires short peptide length Methods employing fluorescently labeled amino acids Fluorophore (conjugated to peptide) [29] Fluorescence SDS-PAGE High/Yes (+) sensitivity (-) involves extra step of fluorophore conjugation Fluorophore (conjugated to amino acid) [26] Fluorescence anisotropy Fluorescence (HPLC)a High/Yes (+) sensitivity (+) high throughput (-) involves extra step of fluorophore conjugation (-) uses aminoacyl-tRNA substrate

aHPLC with a fluorescent readout was used to validate the enzymatic hydrolysis of peptidyl-tRNA while fluorescence anisotropy was used for determining the activity of Pth1.

Table 1: Summary of methods for analysis of peptidyl-tRNA hydrolysis presented here.

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