HCS Products for Nascent Protein Synthesis and Cytotoxicity

Nascent Protein Synthesis and Cytotoxicity

The Click-iT® AHA Alexa Fluor® 488 Protein Synthesis HCS Assay provides a fast, sensitive, nontoxic, and nonradioactive method for the detection of nascent protein synthesis by fluorescence microscopy and high-content screening.

Click-iT® AHA is an amino acid analog of methionine containing an azido moiety. Similar to 35S-methionine, Click-iT® AHA is added to cultured cells, after which it is incorporated into proteins during active protein synthesis. Detection of the incorporated amino acid is achieved via a specific and covalent “click reaction” between an azide and an alkyne, where the azido-modified protein is detected with Alexa Fluor® 488 alkyne.
Click-iT® AHA Alexa Fluor® 488 Protein Synthesis HCS Assay


Figure 1—Dose response curves of nascent protein synthesis using the Click-iT® AHA Alexa Fluor® 488 Protein Synthesis HCS Assay. U-2 OS cells were treated in L-methionine-free media and 50 µM Click-iT® AHA for 30 minutes with cycloheximide (85 pM–5 µM). Cells were washed, fixed, permeabilized, and nascent protein synthesis was detected following a click reaction with Alexa Fluor® 488 alkyne. Imaging and analysis was performed using the Thermo Fisher Scientific Cellomics® ArrayScan® VTI platform.
Product                              Cat. no.
Emission Color*		 Ex/Em†Live‡Fixed§Fixable**
Click-iT™ AHA Alexa Fluor® 488 Protein Synthesis HCS Assay C10289    
350/461
495/519


† Ex/Em = Fluorescence excitation and emission maxima in nm. ‡ Stains live cells. § Stains fixed cells. ** Staining pattern is fixable. NA = Not applicable.