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 usesĀ 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 |