HCS Protein Synthesis Assay

Optimized, automated assays for HCS

The ability to label newly synthesized proteins with both spatial and temporal resolution provides a way to study protein equilibrium and the consequences of its perturbation in disease and in experimental models. Click-iT Plus technology provides a way to label nascent proteins with a range of fluorescent labels, and the opportunity for temporal studies of synthesis and degradation using pulse-chase–type experiments. 

In this example, the Click-iT Plus OPP Alexa Fluor Protein Synthesis Assay Kit provides a fast, sensitive method for the detection of protein synthesis in a HCS format. The assay incorporates O-propargyl-puromycin (OPP) efficiently into newly translated proteins in a complete methionine-containing medium. The protein is then fluorescently labeled with a bright, photostable Alexa Fluor dye in a fast, highly specific, mild click reaction.

Typical protein synthesis assay

Fixed-cell assay protocol.

Imaging mode

  • Widefield or confocal
  • 10x or 20x magnification

Automatically measured properties

  • Fluorescence intensity, morphology, and count values for each object
  • Fluorescence intensity, morphology, and count values of spots in different cell regions
  • Average fluorescence intensity difference and ratios between different regions for each cell
  • Intensity and count ratio between channels within different regions for each cell

Protein synthesis assay images

HCS protein synthesis assay

Hap1 cells treated with cyclohexamide and labeled with Hoechst 33342 and Click-iT OPP followed by addition of an Alexa Fluor 488 azide for chemoselective detection (Click-iT Plus OPP Alexa Fluor 488 Protein Synthesis Assay Kit). The cells were imaged using a Thermo Scientific CellInsight CX5 high-content platform CellInsight CX5 high-content platform (row A) and Thermo Scientific HCS Studio Software, which were used to automatically identify (row B) and quantify the intensity of Click-iT OPP green fluorescence from each cell.

Sample experimental date

HCS Protein Synthesis sample data

Dose-dependent decrease in protein synthesis following treatment of cells with cyclohexamide. The sensitivity of two cell types: wild type Hap1 (red) and Hap1 lacking ATG5 (blue), were compared.