Thermal unfolding data is an easy way to create a “fingerprint” of your protein. By keeping the same ramp rate, you can compare how the unfolding of a single protein changes in response to altering buffer salts, pH, or protein residues. You can also use this fingerprint to compare the differences between two proteins or compare purity in different batches of preparations – variations in the comparison can tell you whether your protein is stabilized or destabilized in response to particular environmental changes.
When looking at large data sets, it is not easy to go through each individual unfolding curve looking for variations. Thus, researchers need parameters that can act as a quick measure of how protein stability has been altered. No parameter is more important for this ranking than the Tm and the melting onset. PR.Stability Analysis software offers quick automated determination of these parameters—but what if you want to confirm the reliability of this data? The software also makes it easy to check these automatically determined values and add or remove markers to improve data analysis. Let’s begin the next installment of Applications Academy: Defining markers in PR.Stability Analysis.
Why are defining markers important?
PR.Stability analysis does an exemplary job determining the melting onsets and inflection points (IPs) using its automatic algorithms. However, data can have errors in it—a capillary may contain a bubble, Region of Interest analysis might determine a subtle Tm not caught by the broad analysis, or you may want to disregard an inflection you know is wrong. Being able to add and remove your own markers is important for in-depth data analysis, as it gives you control over data interpretation.
How does adding and removing markers work in PR.Stability Analysis software?
- Edit markers in specific replicates: It is easy to select and remove markers in the software. The table view makes it easy to find merged sets that do not contain the same marker in all replicates. Expand the merged data to review the markers determined for each set, and then determine whether to add or remove markers.
- Toggle the markers displayed on the graph: Under the display options heading, it is easy to view only onsets, inflection points, or fitted Tm Choose all or none to clean up your presentation view.
- Improved views make finding IPs easier: With the joint display of your ratio (or single wavelength) view and its first derivative in the same window, it is easier than ever to visualize where your IPs occur. Use either window to place your IP markers, and increase your confidence in your conclusions.
How do I add or remove markers in PR.Stability Analysis
A quick guide to editing markers in PR.Stability Analysis
- Your data file will be automatically loaded with the markers determined in ThermControl.
- Review the data table in the window on the left to see which merged data points contain potential outliers. Merged sets that do not show the same IP or onset for all replicates will be displayed in goldenrod.
- Expand the merged data to review individual replicates- use both ratio and first derivative views to determine whether markers need to be added or removed.
- You can also determine true thermodynamic melting temperatures using Region of Interest, which you can learn more about here.
- Use the display options panel on the right-hand side of your graph window to toggle which markers are displayed.
Improve your analysis today with PR.Stability Analysis
NanoTemper’s PR.Stability Analysis software was developed with researchers in mind to help them get meaningful results and conclusions faster. Join the Explorer Community today to get even more out of your Prometheus. Here you can chat with the many users across the globe who use Prometheus to get advanced biophysical information about their proteins and formulations! Plus, find protocols, answers to frequently asked questions, or connect with NanoTemper’s team of applications specialists to get the most out of your Prometheus experience. And stay tuned for upcoming installments in our Applications Academy tutorial series.