Kinomic profiling is achieved using the PamStation®12 Platform Technology (PamGene, International, Den Bosch, The Netherlands) that is a high-content phospho-peptide substrate microarray system. In the PamStation®12 system, active kinases within a cellular/tissue lysate will phosphorylate specific peptide substrates within the microarray which survey the particular kinome (tyrosine or serine/threonine kinome) of interest. These phosphorylation events are detected using fluorescently-labeled anti-phospho-tryosine or anti-phospho-serine/threonine antibodies in real time with kinetic evaluation. Cell lysates are prepared using M-Per lysis buffer with protease and phosphatase inhibitor cocktails (Pierce). The PamChips® are blocked in 2% Bovine Serum Albumin (BSA). Following protein concentration determination (by BCA assay), 5-10 μg of protein for the PTK chip (~1 μg for the STK) will be loaded per well of the PamChip along with standard kinase buffer (Supplied by PamGene), 100 μM ATP, and FITC-labeled anti-phospho-tyrosine (for PTK) or phosphoserine/threonine (for STK) antibodies. For each PamChip®, 4 separate samples can be assayed since each of the wells contains the peptide substrate microarray surveying the tyrosine kinome (PTK) or serine/threonine (STK) kinome. The PamChips® can be assayed 3 chips at a time allowing for 4 samples in triplicate to be assayed at once.
The assay mix is pumped through the PamChips® with a kinetic image capture program (Evolve software, PamGene) in which exposures of phosphorylated peptide substrates are taken as frequently as every 6 seconds for the length of the program (45-90 minutes). The BioNavigator software platform (PamGene) provides a means to analyze the raw data and perform virtually any manipulation of the data. The BioNavigator Microarray software works seamlessly with the open source microarray statistical package, R, commercial products such as GeneSpring as well as knowledge bases such as MetaCore (GeneGo, Inc., a Thomson-Reuters Company), to develop pathway maps and biological networks.