The PamStation is a fully automated CE marked instrument designed for processing a maximum of three PamChip microarrays simultaneously. PamGene’s functional kinase assays are activity-based and detects protein kinase activity directly in cellular and tissue lysates, through measuring peptide phosphorylation by protein kinases.
With PamGene’s kinase activity profiling platform, the activity of kinases in a wide range of cells and tissues can be measured in real time. We use our proprietary 3D peptide microarray technology (PamChip® and PamStation®) which offers a multiplex method for global kinase activity profiling. The assay is very sensitive, requiring only a small amount of lysate to measure the activity of kinases in various samples including cell lines, xenografts and human tissues. Lysates obtained from a few thousand cells can suffice to obtain a kinome profile of the multiple kinases present in these samples. This is accomplished by incubating the sample lysates across peptide substrates immobilized on the 3D surface of the PamChip® microarray (196 protein tyrosine kinase (PTK) or 144 serine/threonine kinase (STK). Kinases present in the lysates will phosphorylate the phosphosites, which are visualized using fluorescently labelled antibodies. Reaction times are typically minutes to an hour with only small amounts of protein input (0.5 to 5 μg per array), making it more sensitive than alternative approaches.
Kinase(s) in the sample (recombinant, cell or tissue lysate) actively phosphorylate substrates on the PamChip®, in the presence of ATP. A FITC-conjugated PY20 antibody, present in the reaction mix is used to detect the phosphorylated Tyr to quantify the phosphorylation signal. Images are taken every 5 minutes to generate real time kinetics data.
Kinase(s) in the sample (recombinant, cell or tissue lysate) actively phosphorylate substrates on the PamChip®, in the presence of ATP. An antibody mix is used to detect the phosphorylated Ser/Thr, and a 2nd FITC-conjugated antibody is used in a detection mix to quantify the phosphorylation signal.
The combined list of detectable kinases can be found here:
Functional assay for determining full length kinase activity.