Procedure:

1. Take out the frozen RNase T1 stock solution and allow it to come to room temperature.
   
   
2. Take 25 test tubes and label them from 1 to 25.
   
   
3. Add increasing volumes of urea stock solution, decreasing volumes of MOPS buffer and a fixed volume of protein stock solution as shown in table 8.1.
   
   
4. Allow the solutions to equilibrate (Note 1).
   
   
5. Switch ON the spectrofluorometer and allow it 30 min warm up.
   
   
6. Set the excitation wavelength to 280 nm and emission wavelength to 320 nm (Note 2).
   
   
7. Measure the fluorescence emission for each of the samples at 90° for 30 seconds (this gives multiple readings, depending on the integration time used for each reading).
   
   
8. Calculate the average fluorescence reading for each of the samples from the multiple readings obtained in step 7 and record them in the observation table (Table 8.1)
   
   
9. Plot the fluorescence emission intensity against urea concentration as shown in figure 8.2.

 

Figure 8.2: A plot between fluorescence intensity against urea concentration showing a typical two-state protein unfolding curve