The core of our technology consists of an array with kinase peptide substrates that are immobilized inside a porous membrane made of aluminium oxide.
PamGene has selected a number of peptides that can serve as substrate for kinases present in the sample (fig A). Functional readout is based on kinetic measurement of phosphorylation occuring on the array using fluorescently labelled anti-phopho-antibodies. These microarrays consists of a porous ceramic membrane (fig B) which allows flow-through of the sample and thereby enhances assay sensitivity. Per array, 144 peptide substrates are immobilised (fig C), and four arrays are combined into a PamChip 4 (fig D). The phosphorylation reaction occurs by an up and down movement of the sample solution through the array, giving the kinases maximal opportunity to phosphorylate the peptides on each array (fig E). When the solution is underneath the array, the CCD camera in the workstation will take an image of each array, which are later used by the software to generate kinetic data curves of each peptide (fig F). The incubation, washing, dispensing of reagents and imaging of the PamChip arrays is done in fully automated workstations called the PamStation 12 (fig G). The data workflow consisting of image quantification, quality control, statistical analysis, visualization and interpretation is performed using the Bionavigator software.
The priciple of our unique flow-through array technology is illustrated here. The pumping of samples through the porous membranes allows faster results and real-time measurements.