The dynamics of global protein synthesis (both spatial and temporal) is an essential parameter to characterize the cellular response under various physiological and pathological conditions. Thus a number of methods have been developed to monitor newly synthesized proteins in cell culture and whole organisms[1].
Ge et al. reported a copper-free alternative to the well-established O-Propargyl-puromycin (OPP)-based monitoring of newly synthesized proteins: The cell-permeable 5’-Azido-puromycin[2] (Fig. 1A) incorporates into the C-terminus of translating polypeptide chains thereby stopping translation.
Resulting C-terminal azide-labeled proteins can be detected via copper-free CLICK labeling that offers the choice to introduce a (Desthio) Biotin group (DBCO-modified (Desthio) Biotin) for subsequent capture and identification tasks or a fluorescent group (DBCO-modified fluorescent dyes) for subsequent microscopic imaging[2] (Fig. 1B).
![Figure 1: 5‘-Azido-puromycin labels newly synthesized proteins in cell culture (adapted from Ge et al. [2]).](/images/ac81846178/nl_5azpuro_0824_1.jpg)
Figure 1: 5‘-Azido-puromycin labels newly synthesized proteins in cell culture (adapted from Ge et al.[2]).
A) Chemical structure of 5’-Azido-puromycin. Visualization of incorporated 5’-Azido-puromycin can be performed via copper-free CLICK-labeling with DBCO-modified fluorescent dyes
B) Nascent protein expression in HeLa cells was visualized by treatment with 10 µM 5’-Azido-puromycin (5Z) with and without pretreatment with 50 µg/ml Cycloheximide (CHX) or 20 µM Bortezomib. Incorporated 5’-Azido-puromycin was subsequently detected by TMR-DBCO or FITC-DBCO, nuclear DNA was stained with Hoechst dye. Scale bar: 15 µm
| Product | Cat. No. | CLICK detection chemistry | CLICK detection reagents |
|---|---|---|---|
| 5’-Azido-puromycin | CLK-110 | Copper-free | DBCO-modified fluorescent dyes DBCO-modified Biotin |
| O-Propargyl-Puromycin | NU-931 | Cu(I)-catalyzed | Azides of fluorescent dyes Azides of Biotin |
[1] Tang et al. (2023) Nascent Proteomics: Chemical Tools for Monitoring Newly Synthesized Proteins. Angew. Chem. Int. Ed. 62:e202305866 (1-11).
[2] Ge et al. (2016) Puromycin Analogues Capable of Multiplexed Imaging and Profiling of Protein Synthesis and Dynamics in Live Cells and Neurons. Angew. Chem. Int. Ed. 55:4933.
