Table 2.

Comparison of methods for probing translation.

	Puromycin reagents	Non-puromycin alternatives	Method of detection	Comments
Translation rate measurement	Radioactive puromycin [35]	Radioactive amino acids (AAs) [55]	Scintillation or autoradiography	AA analogs do not terminate translation, but puromycin can be used without predepletion of endogenous AAs
	Puromycin/OPP (SUnSET) [8], [22]	Clickable AAs [56]	Immunoblot or FACS
Visualization of newly synthesized proteins	Puromycin/OPP (RPM, Puro-PLA) [8], [14], [42]	Clickable AAs (FUNCAT, FUNCAT-PLA) [42], [57]	Immunofluorescence	AA analogues do not terminate translation, but only puromycin can be used to label sites of active translation when combined with inhibitors of elongation
Translatome analysis	N-blocked biotinylated puromycin (RiboLace) [16]	Deep sequencing of nuclease-resistant ribosome-protected mRNA (ribo-seq) [58]	Next generation sequencing	RiboLace can be used to affinity purify translating ribosomes prior to ribo-seq analysis
	Biotinylated puromycin/OPP (PUNCH-P/OPP-ID) [44], [47]	AA isotopes (pSILAC) [59] Clickable AAs (BONCAT/QuaNCAT) [56], [60]	Mass spectrometry	AA analogs do not terminate translation, but puromycylation is rapid, AA nonspecific and can occur on isolated ribosomes