Zoom in on your results using Regions of Interest in PR.Stability Analysis software

Data analysis is a challenging process—researchers have mountains of data to go through—and expediting analysis with automated software solutions is a crucial tool to save you time and effort. However, no algorithm can replace the human eye when it comes to double-checking the results. So how can researchers save time with data processing but also feel confident in their conclusions?

PR.Stability Analysis software has a great new feature—Region of Interest—to help you develop that much-needed confidence in your conclusions. This feature enables you to focus only on the portion of the data that is important to you, and the fitting algorithms are re-tooled only for the portion of the data you highlight. Catch subtle transitions, quickly view and rank the parameters of interest important to you, or focus only on a specific domain of your protein. Welcome to another installment of Application Academy: How to Use Regions of Interest.

 

Why is the Region of Interest important?

Until now, Prometheus users processing data using PR.ThermControl or PR.ChemControl software have been applying the inflection point (IP) and onset algorithms to their full data set across the complete temperature or chemical gradient. But many proteins do not exhibit unfolding until quite a way through the gradient. There can be issues with sloping baselines that convolute analysis. Perhaps bubbles sneak into your sample. Or maybe you only use one domain transition to draw conclusions about your experiments. The region of interest feature allows you to select only a portion of the gradient to analyze so you can determine true thermodynamic parameters from your data. Improve your data analysis while focusing only on the information you care about.

 

How does Region of Interest work in PR.Stability Analysis software?

  • Select only the portion of the unfolding curve you are interested in: There are many reasons to focus on a limited window of your data—for examining only one domain unfolding, when trying to determine if a region contains a subtle unfolding transition, or for getting a better fit due to sloping baselines. The Region of Interest feature allows you to consider these examples and select only the data that matters to you.
  • Extract thermodynamic parameters from your PR data: Smaller transitions can be missed with the IP determination by ThermControl software. Using the region of interest allows you to calculate Tms for subtler transitions, and ends tedious manual evaluation of each replicate.
  • Options for two- or three-state fit: Fine tune the fitting algorithms to feel confident in your conclusions.
  • Easily highlight and view the region of interest on your graphs: PR.Stability Analysis’s interface makes it easy to see where you have placed your Region of Interest within the full data set, and is easy to adjust the selection.

 

How do I create a Region of Interest in PR.Stability Analysis?

Check out this video with NanoTemper Application Specialist Lindsay Dawson to see how easy it is to define and analyze a region of interest in your dataset.

 

A quick guide on using Regions of Interest

  • Above your chart displays, select the menu for “region of interest”
  • You can apply a two- or three-state fit, or if you have a reference sample you can examine the similarity
  • A window will open in the lower-left corner showing the temperature range and the number of samples evaluated.
  • On the graph, you should see the Region of Interest highlighted. You can toggle this on/off by clicking the eyeball under the Region of Interest window.

 

Improve your analysis today with PR.Stability Analysis

PR.Stability Analysis is designed to enable our Prometheus users to get highly precise information about the data that is most important to them. To get even more out of your Prometheus, sign up for the NanoTemper Explorer Community and chat with other NanoTemper users. Plus, you can get your questions answered from our team of dedicated Applications Specialists. You can also find great tips on PR.Stability Analysis software, various protocols, and even fun facts! Comment to let us know what other topics you’d like to see, and stay tuned for additional tips and tricks on PR.Stability Analysis and more in our Analysis Academy series.

About the Author

Stefanie Kall is an Applications Specialist with NanoTemper Technologies. She received a biochemistry PhD from University of Illinois in Chicago for her work characterizing inhibitors for kinases via X-ray crystallography. She also has a MSc in Biotechnology from Northwestern and a BSc from (The) Ohio State University in Biochemistry. When she is not training people on NanoTemper instruments, she enjoys running, baking, and playing video games.
Stefanie Kall