During data analysis you will perform these major steps:
- Blank and normalize your fluorescence data
- Fit the data directly in GraphPad Prism to determine:
- the ΔGunfolding
- the CM
- Manually manipulate the fluorescence data to create two plots:
- fraction of folded protein (Xf) vs. [urea]
- ΔGunfolding vs. [urea]
Part 1: Blank and normalize your fluorescence data
- The following video helps get you oriented to your data and demonstrates how to average your blanks and subtract them from your experimental values. NOTE: For all videos, you should be able to actually read the numbers being shown. You may need to view full-screen, and/or manually increase the video quality. You can also turn on closed captioning for all videos.
- NOTE: The urea concentrations tested are included in the “Setup” page of the data file. These may not be the same concentrations you were planning to use! Be sure to take the urea concentrations from the data file, not from your plan.
- For each protein (wt and mutant), create plots that overlay the emission spectrum of your protein at each urea concentration, as demonstrated in the following video.
- The plate reader records values in relative fluorescence units (RFU), which depend on the instrument settings. All samples should have the same protein concentration, but pipetting errors can cause slight (or large) variances. Therefore, it is helpful to normalize the data, using the fluorescence at a wavelength that is not very affected by the state of the protein (340 nm) as a reference point. First, look at the plots you created in step 2 and be sure the folded curves have a peak around 324 nm, while the unfolded curves shift to higher wavelengths. If not, talk to the teaching staff before proceeding with the data analysis.
- Normalize the data by calculating the ratio between two emissions: 324/340. 324 nm is the point where you should observe a peak in the folded protein curves. 340 nm is the approximate point where all the curves come closest to intersecting.
- Discard data from any wells that appear to have no (or too little) protein. These would appear as approximately a straight line in the fluorescence vs wavelength plot, and/or give a negative or otherwise unreasonable 324/340 ratio. DO NOT use data from these wells in any of the remaining analyses.
Part 2: Fit the data directly in Prism to determine the ΔGunfolding and CM
- This video walks through the basics of fitting data in Prism. Download the sample data file so that you can follow along with the video. Sample file can be downloaded from Canvas here: Sample Lab 9 Data Analysis File
- PLEASE NOTE: This video was made using Prism 7 for Mac. Your version may differ slightly.
- The first thing you need to do is download the correct fitting equation from the sample data file. Unfortunately you can’t directly download a Prism equation, but this is a workaround:
- Open the sample file and navigate to the “Nonlinear fit of…” page under the “Results” heading.
- Loo