Cellular Phosphorylation Assays
Protein phosphorylation, principally on serine, threonine or tyrosine residues, is one of the most important post-translational modifications, and the most common modes of regulating protein function. In most cases, the protein will switch between a phosphorylated and an unphosphorylated form under kinase function. Radiolabel studies indicate that approximately 30% of proteins in eukaryotic cells are subject to phosphorylation. Protein phosphorylation plays key roles in the regulation of many cellular processes including cell cycle, growth, apoptosis, differentiation, metabolism and signal transduction pathways. Abnormal protein phosphorylations in some cases are implicated in many disease states such as cancer, diabetes, and inflammatory disease.
By using in vitro biochemical kinase assays such as the typical sandwich ELISA, researchers can perform hypothesis testing and drug screening, however, it cannot represent the intracellular environment. Cell-based Phosphorylation Assays offer the advantage of physiological ATP concentrations at the site of action and the phosphorylation of physiological substrates. Analyzing protein phosphorylation within intact cells may more accurately replicate the status of specific signaling networks.
Creative Bioarray is experts of cell based kinase assays and Phosphorylation Assays. We are confident, especially in the field of drug development of small molecule kinase inhibitors. Creative Bioarray can help you to determine compounds ranking based on cell based phosphorylation data which are critical for the decision on preclinical testing. After in vitro cell based kinase profiling of your compounds, a similar experimental designing based on cellular phosphorylation assays will substantially increase your knowledge of your kinase inhibitors.
With 50 Kinase cell lines available, Creative Bioarray provides several different cellular phosphorylation assays to accelerate your research of kinase functional activity, dimerization, characterization, and inhibitor screening. We provide technical methods of ELISA or Western blot based on the customer’s requirements.
Check Creative Bioarray available cell line here.
- Western blot: It uses gel electrophoresis to separate native or denatured proteins by length of the polypeptide. Proteins are then transferred to a PVDF or nitrocellulose membrane, and then a phospho-specific antibody can be used to identify the protein of interest.
- ELISA: This format uses a capture antibody specific for the desired protein, independent of the phosphorylation state. The target protein is added allowing it to bind to the antibody-coated plate. A detection antibody is then added that will specifically bind to the phosphorylation site of the target protein. The results are analyzed by colorimetric or fluorometric detection.
Figure 1. Detection of Phosphorylation by WB
Figure 2. IC50 values for two experimental compounds by ELISA tests
- IC50 values are determined by testing 8 compound concentrations in semi-logarithmic steps (each concentration in duplicates).
- Quality assurance is provided by calculation of Z' factors for Low/High controls on each assay plate and by including a full IC50 curve for a reference inhibitor to monitor adequate dose/response relation in your assay run.
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