Click-&-Go® Plus 594 OPP Protein Synthesis Assay Kit enables fast and sensitive imaging of nascent proteins in complete, methionine-containing media. OPP (O-propargyl-puromycin) is an analog of puromycin that contains a terminal alkyne group, enters the acceptor site of ribosomes and incorporates into nascent polypeptide chains. Unlike HPG or AHA labeling reagents OPP is not an amino acid analog; thus, OPP can be added directly to cells in complete media (i.e., methionine-containing) or used to detect in vivo protein synthesis. In the next step, OPP-labeled nascent proteins are imaged through a chemoselective ligation, or “click” reaction, between the red fluorescent AZDye 594 Azide Plus (Alexa Fluor 594 ® equivalent) and the OPP-alkyne allowing the modified proteins to be detected by imaged-based analysis. The kit contains all of the components needed to label and detect the incorporated OPP in newly translated proteins in samples of adherent cells. The kit includes sufficient reagents for the labeling of 25 18 mm × 18 mm coverslips using 1 mL of reaction buffer per test.
Although protein synthesis is a conserved and essential cellular function, it is often regulated in a cell-type-specific manner to influence cell fate, growth and homeostasis. Most methods used to measure protein synthesis depend on metabolically labeling large numbers of cells with radiolabeled amino acids, stable isotope-labeled amino acids, bioorthogonal noncanonical amino acid tagging (L-azidohomoalanine or homopropargylglycine or their combination). Because these methods typically depend on specialized growth conditions, they have been largely restricted to yeast, bacteria and cell lines. Application of these techniques for investigating protein synthesis within mammalian systems in vivo has been challenging.
The use of O-propargyl-puromycin (OPP), an analog of puromycin that contains a terminal alkyne group, has facilitated the quantification of protein synthesis within individual cells in vivo. OPP enters the acceptor site of ribosomes and incorporates into nascent polypeptide chains. Unlike traditional methods mentioned above, OPP is not an amino acid analog; thus, OPP can be added directly to cells in complete media (i.e., methionine-containing) or used to detect in vivo protein synthesis. It also can be used with cell lines that are sensitive to media exchanges or incubation in methionine-free media. The combination of high cell permeability and signal-to-noise ratio makes OPP an ideal candidate compound to study nascent proteomes across a wide array of cellular types and conditions. The kit contains all of the components needed to detect incorporated OPP with red-fluorescent AZDye 594 Azide Plus (Alexa Fluor® 594 equivalent), and blue-fluorescent Hoechst 33342 dye for nuclear staining. A sufficient amount of reagents is provided for imaging 25 coverslips or 250 wells using 96-well plates.
An alkyne analog of puromycin that is efficiently incorporated into newly translated proteins in complete methionine-containing media.
A water-soluble ligand for copper-catalyzed azide-alkyne cycloadditions.
Excitation maximum: 494 nm
Emission maximum: 517 nm