Samples being placed in the NanoPro for assay.
The Translational Research Technology (TRT) unit has purchased a NanoPro 1000 system (Protein Simple) to measure protein biomarkers. Rather than separating the proteins by size as in Western blots, the NanoPro system separates proteins by their isoelectric focusing point. The technology is primarily applied to evaluating post-translational modification of protein biomarkers. Detection and quantification of the phosphorylated forms of signaling kinases is an example of an application, but other applications are available. The system is a fully automated medium throughput instrument, which limits variability and generates results that are more reproducible from run to run, and over time.
The NanoPro system utilizes microfluidics, therefore very small sample amounts are needed. This technology is ideal in a setting requiring multiplex measurement of small biospecimens. The NanoPro has the ability to assay up to 96 wells, meaning that this technology can perform any combination of 1-8 targeted biomarkers and between 12-96 samples per run (ideally samples are run in sets of 12).
Researchers can now use nanoscale methods to identify and characterize biologic signatures and measure targeted post-translational biomarker response to intervention. Various signaling assays are currently available. TRT will work with investigators to develop new applications and targets.
Above is an example of an experiment in which fibroblast-like cells were initially silenced for either MKK4 or MKK7, kinases upstream of JNK. Cultures were then challenged with either IL-1β or media alone. The signature of the substrate kinase, JNK, was measured on the NanoPro. Each silenced kinase is compared to stimulated non-silenced and media controls. Both the upstream kinases are depicted. The peaks at different pI's reflect different isoforms and phosphorylation states of JNK. The differential kinase activity of MKK4 and MKK7 is different in the chromatogram.