Assessing kinase activity within biological samples is quite challenging.
Commonly this is done by using in vitro assays where immunoprecipitated kinases are incubated with an exogenous substrate in the presence of ATP.
The phosphorylation status of the substrate can be further assessed by colorimetric, radioactive or fluorometric assays.
Pamchip peptide array technology allows the parallel measurement of multiple kinase activities, both in cells and in-patient samples, by profiling peptide phosphorylation changes using a pharmacology-on-chip approach.
Pamchip arrays consist of a highly porous ceramic membrane, on which 144 Serine/Threonine kinases (STK assay) or Tyrosine kinases (PTK assay) peptide substrates are immobilized, with each Pamchip containing 4 individual arrays.
Measurement of the activity of the kinases present in the sample of interest is based on the quantification of peptide phosphorylation.
Fluorescently labelled anti-phospho antibodies are used to detect phosphorylation intensities of the different immobilized peptides.
The fluorescence intensity data of phosphopeptide signals of control and treatment groups are uploaded into the Bio-navigator software, which uses statistical tests and algorithms for inferring and scoring upstream kinase analysis associated with the Pamchip phosphopeptide profile of interest.
Outputs of this analysis are compatible with Ingenuity or Metacore pathway analysis software and predict the downstream biological effects or calculate the pathway specific activation or inhibition Z-scores.
The ranked kinome activity signature associated with a Pamchip phosphopeptide profile of interest can then also be projected onto the phylogenetic kinome tree.