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Assay Tutorials : May 1, 2008 ( )
Site-Specific Phosphorylation Profiling
Ti-Tyr Chips Deliver Quantitative Analyses
Site-specific tyrosyl phosphorylation is a key mediator of intracellular communication and represents a rapid readout of the cell-signaling network’s response to drugs or other stimuli. For this reason, proteins involved in these signaling responses have become major targets for therapeutic intervention. The complexity of these responses, however, requires powerful tools that can monitor information flow through the signaling network and identify important biomarkers associated with disease or drug responses.
Epitome Biosystems developed quantitative phosphorylation-profiling chips that broadly survey the signaling network. The company’s approach provides site-specific phosphorylation data, utilizes quantitative reference standards, is multiplexed, and accommodates parallel processing of multiple samples.
The EpiTag™ assay platform measures peptide fragments as surrogates for proteins, making it possible to develop accurate reference standards using synthetic peptides. The approach allows for simultaneous measurement of multiple phosphorylation sites on the protein as well as multiple sites of phosphorylation across different protein targets.
The Ti-Tyr™ Profiling Chip is a tyrosine-phosphorylation focused product based on the EpiTag technology. Tyrosine phosphorylation is measured quantitatively at a site-specific level after proteolytic fragmentation of samples. The Ti-Tyr chip allows for multiplexed analysis across more than 75 phosphotyrosine sites on 62 different proteins using a planar microarray.
Cell-Signaling Network Dynamics
EGF-stimulated A431 cell lysates were analyzed using the Ti-Tyr chip. Cells were grown to 80% confluence, serum starved for 24 hours, and treated with EGF (100 ng/mL) alone or EGF after pretreatment with SL327 (a MEK inhibitor) for eight minutes. Lysates were harvested, digested, and analyzed on Ti-Tyr™ Chips. The concentration of each target was determined by interpolation from standard curves.
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