Click-iT™ HPG Alexa Fluor™ 488 Protein Synthesis Assay Kit (Invitrogen™)

The Click-iT™ HPG Alexa Fluor™ 488 Protein Synthesis Assay Kit provides a fast, sensitive, non-toxic, and non-radioactive method for the detection of nascent protein synthesis utilizing fluorescence microscopy, high-content imaging, or flow cytometry. Included in the kit are L-homopropargylglycine (HPG), an amino acid analog of methionine containing an alkyne moiety, and Alexa Fluor™ 488 azide. The HPG is fed to cultured cells and incorporated into proteins during active protein synthesis. Addition of the Alexa Fluor™ 488 azide leads to a chemoselective ligation or 'click' reaction between the green fluorescent azide and the alkyne, allowing the modified proteins to be detected by imaged-based analysis.

• Non-radioactive alternative—an alternative to the traditional ³⁵S-methionine
• Visualize bulk protein dynamics—fluorescent tagging of proteins allows their localization to be determined, including aggregation
• Specificity—selective, specific reaction between label and detection tags
• Stability —product contains an irreversible, covalent bond
• Multiplex-enabled—use in conjuction with Click™-iT AHA (azide amino acid and alkyne dye) to detect spatial and temporal differences
• Applicability to biological samples—easy detection; high sensitivity and low background, regardless of complexity

The Click-iT™ HPG Alexa Fluor™ 488 Protein Synthesis Assay Kit has been successfully tested in HeLa, A549, and U-2 OS cells with a variety of reagents that inhibit protein synthesis, including cycloheximide and anisomycin. The applicability of these probes to monitor protein degradation has also been shown using inhibitors of the proteasome (MG132 and Bortezomib) and blockers of autophagy (chloroquine) in HeLa cells.

Additionally, due to differences in Click-iT™ chemistry between the Click-iT™ HPG Alexa Fluor™ 488 Protein Synthesis Assay Kit and Click™-iT AHA with Alexa Fluor™ 594 Alkyne, these reagents can be used in conjunction for spatial or temporal determination of differences in nascent protein synthesis.