The processes of cellular proliferation and the progressive acquisition of a specialized phenotype are controlled by highly coordinated mechanisms. Evidence from several studies suggest that polypeptide growth factors responsible for normal growth and differentiation of tissues can contribute to neoplastic transformation. These polypeptide growth factors exert their biological effects by binding to high affinity membrane bound receptors on target cells. Several growth factors (e.g. FGF, EGF, IGF etc.) bind to cell surface receptors possessing protein tyrosine kinase (PTK) activity. The activation of PTK activity by these mitogenic hormones has been found to contribute to the growth and transformation of several tissues.
My doctoral studies involved the development of peptides that could cause the down-regulation of the EGF receptor associated tyrosine kinase activity. For these studies I used the "non-radioactive Tyrosine Kinase Assay Kit" from Roche Molecular Biosystems. This kit utilizes a specific peptide substrate that is biotinylated at the amino terminus. The enzyme reaction is quenched by the PTK inhibitor piceatannol. The phosphorylated and dephosphorylated substrates are then immobilized on stretpavidin coated microtiter plates and the reaction mixture is washed out. The fraction of phosphorlylated substrate is detected immunochemically by a highly specific antibody conjugated to peroxidase. The rate of dephosphorylation can be quantified by the use of a standard phosphopeptide, provided in the kit.
The biggest advantage of this kit is that it is non-radioactive, hence does not require tedious procedures associated with handing of radioactive substances. The sensitivity of this kit is comparable to the commonly used radioactive assays. The kit gives a low background and is very sensitive (as well as specific) for the measurement of PTK activity. The kit does not cross react with tyrosine, phosphoserine, or phosphothreonine. It includes a phosphopeptide standard and for exact quantitation of tyrosine kinase activity. The calibration graph generated by this phosphopeptide is highly reproducible in each experiment. The assay is designed in the ELISA format which is easy to perform and interpret.
The kit contains a 96 well plate format to perform the assay. If the phosphopeptide is used everytime to generate the standard graph, it is not possible to do too many samples (assuming that each sample is at least in duplicates), since only one streptavidin coated plate is provided in the assay. In that sense, the assay kit is quite expensive. Moreover, the detailed working procedure (mentioned in the specification sheet) is not very clear - I had to read it several times to comprehend it. The flow charts and tables are also not very clearly described. The working protocol emphasizes the importance of a positive control (e.g. EGFR from A431 cell membranes) however this positive control is not provided in the kit. The kit also does not include Mg2+/ATP, which is crucial for performing the assay. The problem lies that these points are not mentioned in the relevant page of the catalog (page 371, 2000 biochemicals catalog), but are mentioned in the literature accompanying the kit, which is extremely misleading. The technical support team also does not enlighten you of the fact that you need to order some additional chemicals, which are not provided in the kit.
Apart from these concerns, I was totally satisfied with the performance of the kit. The tyrosine kinase assay kit provides a fast specific and reliable non-radioactive method for both the quantitative and comparative determination of soluble as well as receptor-associated tyrosine kinase activity in cells.
Piyali Dasguptam
Postdoctoral Research Fellow
Department of Pathology
College of Physicians and Surgeons (14-460)
Columbia University, New York
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