Errors in cell-signaling pathways play a central role in many diseases including cancer, diabetes, and autoimmune disorders. The ability to study these signaling pathways and accurately measure protein and protein phosphorylation levels is increasingly important in drug discovery and the development of disease models.
Traditional multiplexed immunoassays such as those conducted on the Luminex® xMAP® platform have advanced our understanding of intracellular communication by enabling the ability to measure multiple total or phosphorylated sites along a signaling pathway in a single sample, and simultaneously measure sites in multiple pathways. However, multiplexing still presents some challenges as there is a lack of phosphospecific antibodies that work well on the Luminex xMAP platform due to:
- The requirement for native protein confirmation instead of recognition of denatured proteins on Western blots
- Complex buffer interactions with phosphoproteins
- Matrix and inter-antibody effects when multiplexed
Resulting difficulties include a lack of sufficient targets for pathway analysis, qualitative or relative quantitative results, and the need for separate wells to analyze each phosphorylated site on a specific analyte as well as total protein.
To overcome these challenges, the MILLIPLEX® MAP EpiQuant™ receptor signaling portfolio (EMD Millipore) enables researchers to measure phosphorylation levels of multiple sites, as well as total protein, simultaneously with absolute quantitation.
Based on the Luminex xMAP platform, the technology uses antibodies against defined protein sequences to capture peptides resulting from protein linearization and digestion in a single cell lysate sample. The fragments generated are similar to those generated using mass spectroscopy. These same peptides are then used as standards to generate curves with picomolar sensitivity.
This multiplexing technology provides faster answers to cell signaling questions compared to traditional Western blots, mass spectrometry analyses, and radioactive phosphorylation assays requiring large amounts of resources, time, and sample.