Protein synthesis is a strictly regulated process and many critical controls on gene expression occur at the translational level to ensure that the production of specific cellular proteins is turned on / off rapidly under specific conditions (heat shock, starvation, etc.). It is essential in cell growth, proliferation, signaling, differentiation, or death; therefore, the identity and quantity of proteins synthesized are critical in determining the physiological state of the cell.
Methods for detecting and characterizing nascent proteins, or changes in spatial and temporal protein expression/degradation patterns during disease, pharmacological treatments, or environmental changes are important for the evaluation of cytotoxicity.
Biovision’s EZClick Global Protein Synthesis Assay Kit uses a novel chemical method based on an alkyne analog of puromycin, O-propargyl-puromycin (OP-pure). Pure-OP stops translation by forming covalent conjugates with nascent polypeptide chains. Truncated polypeptides are rapidly converted by the proteasome and can be detected based on a click reaction with fluorescent azide (Ex / Em = 494/521 nm).
OP-pure does not require methionine-free conditions and can be used to label nascent proteins directly in cell culture. We provide materials sufficient for 100 simple and specific assays to detect nascent proteins synthesized under various physiological conditions in real-time, and cycloheximide, a protein synthesis inhibitor that serves as a control.
Glycans are vital components of glycoproteins, glycolipids, and proteoglycans in all domains of life. Glycosylation occurs co- or post-translationally in> 50% of eukaryotic proteins, resulting in secreted, intracellular, or membrane-associated glycoproteins that are crucial in cellular processes, protein bioactivity, and metabolic turnover.
Intracellular glycans mediate protein folding, stability, and trafficking, while at the cell surface they participate in recognition, cell-cell interactions involved in embryonic adhesion, migration, and development; host-pathogen interactions critical for bacterial and viral infections; and initiation of the immune response. Aberrant glycosylation profiles correlate with inflammation and are a universal feature of cancer, with sialic acids playing an especially prominent role as tumor-associated carbohydrate antigens (TACA).
Altered sialylation of tumor cell surfaces is associated with several critical malignant properties including invasiveness and metastatic potential, suggesting its implication in clinical diagnosis. Since glycoproteins are not directly encoded in the genome, glycoprotein characterization and analysis methods are of great interest.
Therefore, BioVision offers the EZClick Sialic Acid Modified Glycoprotein (ManAz) Assay Kit, a highly specific, simple, and robust method for labeling and detecting N-linked glycosylation of cell surface proteins. We use a modified mannosamine precursor that is fed directly into cells, converted to sialic acid by the sialic acid biosynthetic machinery, and transported to the Golgi apparatus for the manufacture of glycans.
Followed by a click reaction with an alkyne-containing dye, this system offers a powerful method for imaging the location, traffic, and dynamics of glucans, or FACS detection for quantitative studies. Labeled glycoproteins can be detected directly on 1D or 2D gels using appropriate excitation sources, or enriched by immunoprecipitation with biotin-alkyne or antibodies prior to proteomic analysis. We provide sufficient materials for 100 assays in a 96-well plate format.