The UCeP assay is conducted using living cultured cells at human physiological temperatures, making its results more relevant to human biology than traditional in vitro biochemical assays that use affinity capture. As a result, you’ll obtain insights that more accurately reflect the true interactions between your test drug and its targets in human cells. Moreover, some protein targets are structurally unstable at elevated temperatures, rendering them unresponsive in thermal shift assays. Unlike these assays, the UCeP assay operates across various denaturant concentrations, not varying of temperature points. From our in-house UCeP studies, UCeP has been successfully demonstrated to capture targets that are not responsive to thermal shift assays. Overall, UCeP is a robust technology for validating your drug’s putative targets and identifying those that are elusive to any kinds of thermal shift assays.

Although we have not yet tested the UCeP assay in our lab, based on our understanding of other biophysical assays, it is technically compatible with various type of biological sample. Given the homogeneity of cell lines, we prefer to perform UCeP assay using cell lines in our lab. These settings of UCeP assay have been demonstrated robust performance in our benchmarking experiments. However, for highly heterogeneity samples like tissues and spheroids, additional optimizations and microdissection may be necessary for better accuracy. Despite these challenges, we are keen to tailor our UCeP assay for your tissue samples to obtain critical information, that can push your research forward.

In UCeP assay, most drug bound proteins exhibit either regular or irregular bell-shaped abundance ratio curves. These curves span the x-axis from 0M to the highest molarity, denominated by the relative abundance of vehicle. Unlike sigmoidal curve, we do not measure the half-maximal point of the bell-shaped curve. Instead, we simply compute the area under the curve (AUC) to represent the magnitude of stability change of drug bound proteins. To improve throughput and sensitivity of our assay with mass spectrometry, we selectively focus on 4 or 5 denaturant points that frequently yield the highest readout for analysis. We calculate average abundance ratio (AAR) from these denaturant points to rank target proteins in increasing orders. A higher AAR indicates stronger binding affinity to test compounds, which should be further validated through dose-response study. Given the nature of UCeP technology and fewer data points for each protein, we present the AAR data using scatter plots to visualize the stability changes of whole proteome between 2 biological replicates. Ideally, your protein targets should consistently exhibit high AAR values across these replicates with good correlations between 2 replicates.

In our dose-response study, UCeP can detect drug binding affinity down to nano molar range. However, for assessing drug target selectivity, we recommend dose treatment between 1 and 10 μM. This strategic approach enhances the signal from off-targets that have lower affinity for the drug. Additionally, higher dosing ensures rapid effective dose accumulation in cells when treating the cells for 5 or 10 minutes during direct targets assessments. As a result, you gain a broader view of the on/off-target profile of your test compound at the most effective dosing level. In a standard UCeP-ID assay, we may request you to provide us enough test compounds for two replicates of experiments. For instance, if your stock compound concentration is 10mM, we would need at least 25 μL from the stock.