Lab 11: DATA ANALYSIS
During data analysis you will calculate the following:
- The KD for binding of DNSA to WT HCAII
- The KD for binding of DNSA to your HCAII mutant
- The KD for binding of AZ to WT HCAII
- The KD for binding of AZ to your HCAII mutant
For these calculations, you will use Excel to determine values and then plot ligand concentration versus fraction bound in Prism.
KD for binding of DNSA to HCAII
Step 1: Look at your data. You should have fluorescence emission data at 470 nm (F470) at various concentrations of DNSA that has been added to a solution containing HCAII. You should also have the volume of solution in each well, and the total concentration of DNSA ( [DNSA]total) for each point in the titration. Identify any outliers in your data and determine whether they can be removed by applying the Dixon’s Q test.
For help with statistics, see the Appendix (link opens in a new tab): Appendix 2: Statistical methods used in 551 data analyses
The following video will help you become oriented to your data and show you how to identify and remove outliers.
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 quality using the button in the lower right corner of the video. You can also turn on closed captioning for all videos.
Step 2: Correct the F470 data for dilution during the titration (i.e. determine Fadjusted). Use the following equation:
Step 3: Calculate the fraction saturation, r, at each point (i.e. normalize the data). Use the following equation:
where Fmin is the smallest value of Fadjusted and Fmax is the largest value of Fadjusted.
Here is an example wt data set that has been analyzed through step 3. Note that the r values range from ~0 (at low DNSA) to ~1 (at high DNSA).
Step 4: Calculate the concentration of free DNSA at each point (i.e. calculate [DNSA]free). Use the following equation:
The concentration of [HCAII]total is probably 0.25 μM, unless you used something different.
Step 5: Use Prism to plot r as a function of [DNSA]free. Note that even though you did the experiment in triplicate, each Y-point (r) has a different X ([DNSA]free), and so you cannot enter them as replicates in Prism. You can still enter all the data into Prism at once, you will just enter one “replicate” each for wt and mutant that contains all the data.
When you open Prism, choose “Enter and plot a single Y value for each point” from the initial menu. Enter the [DNSA]free and r values for wt. Then add the mutant values below, always keeping [DNSA]free in the “X” column but using two separate columns for r wt and r mutant.
See example:
The plot of your data should look hyperbolic, as is expected for ligand binding behavior.
See example plot:
Step 6: Fit the data to the Langmuir isotherm to determine the KD.
Fitting the equation should give you the KD. You can also use Prism to determine whether there is statistical difference between wt and mutant. The following