Question

Give one example of how you would assay for overexpression of RTK (Receptor Tyrosine Kinase). Would it be an immune complex kinase assay then determine if it's a tyrosine kinase? I'm not sure.

Answer 1

Answer – Beside the classical way of detecting using the Immunoprecipitation methods, the Ag –Ab based methods for detecting the protein - protein interaction may be useful for the accessing the overexpression of RTKs. The immune complex assay is another option for the detecting the overexpression of protein kinase. In this assay the TPKs are immunoprecipitated, allowed to auto phosphorylate in the presence of labelled ATP, run out on an SDS-PAGE gel, and detected by autoradiography .The alternate way is to access the functional state of RTKs by providing the potential substrate along with the labelled ATP in reaction mixture . There are two ways for evaluating this Ist , the exogenous substrate is a protein (enolase or α-casein), permitting simultaneous assessment of auto phosphorylation and exogenous substrate phosphorylation secondly by utilizes a peptide substrate, resulting in a rapid, high-throughput assay that evaluates only exogenous substrate phosphorylation.

Beside this method we can use the ELISA based kit method (It is reported in a journal and can be useful so, mentioned here), and has the rapid, sensitive method to check the activity and so detects the substrate specificity in cell and tissue extracts. The sensitivity is can be detected using the microgram and Nano gram amounts of total protein extracted from the sample. The K-LISA™ PTK Screening Kit is a useful tool for studying kinase inhibitors and mutational effects or for rapid assessment of PTK activity in a variety of sample types. The kit features a multiplex array of three synthetic, random polypeptide substrates of glutamic acid, alanine and tyrosine, which allow comparison of specificity differences among PTKs.