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.

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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

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

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.

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HCS reagents optimized for use with your HCS platform