Troubleshooting.

Step	Problem	Solution
1	The baseline is not flat.	Run a spectrum without a cell to make sure the baseline with N2 is flat. Clean the cell as described in previously4.
2	The protein sample has very low ellipticity between 250 and 200 nm,	Check that the protein concentration is correct as described previously4
3	There is no change in ellipticity upon mixing the ligand or titrant with the protein.	Not all ligands will become optically active or change the conformation of a protein upon binding. In addition, some proteins are resistant to denaturants such as urea. If this is the case, check if the addition of ligand or titrant changes the stability to thermal denaturation and use this method to determine the binding constants or free energy of folding47
	The ellipticity changes as a function of time.	The protein may equilibrate very slowly. Try preparing samples with various concentration of ligand or titrant and incubate overnight. This uses more protein solution that serial additions but may solve the problem.
5	The change in ellipticity does not plateau, when corrected for the ellipticity of ligand, even at very high ligand to protein ratios.	The concentration of protein in the pure solution and in the solution containing ligand plus protein may not be equal. If one is using Teflon stoppered cells and mixing by inverting the cells, the solution may be leaking. Try redoing the titration at lower concentrations in a cell that is stirred with a magnetic stirring bar. Alternatively, the protein-ligand or protein in the presence of titrant may not be at equilibrium. Try incubating the mixtures for longer periods of time.
6	The ellipticity of the ligand is not linear function of its concentration.	The ligand may self associate or may have too high and absorbance. Make sure that the dynode voltage is below 5004. Try titrating samples of a fixed concentration of ligand with increasing concentrations of protein, if the ellipticity of the protein is linear as a function of concentration.
10	Plots of the free energy of folding are not a linear function of denaturant or osmolyte.	There may be unfolding intermediates or the samples may not be at equilibrium. Try incubating the samples for a longer period of time before obtaining the spectra. However in some cases the changes are intrinsically non-linear and require more complicated analyses11,12,22
11	The ellipticity change saturates at a low ligand to protein ratio, but the data cannot be fit by equation (23) using non-linear least squares curve fitting programs.	First, make sure that the initial parameters for maximal change, protein concentration and KD are close to what would be expected from the raw data. If changing the initial parameters does not work, the protein may have more than one class of tight binding sites and the binding may be cooperative and require more complicated approaches to describe the binding curves30,37,48.