Troubleshooting

Problem	Potential Cause and Solution
A significant decrease in fluorescence is detected in the buffer reference titration	This can occur for several reasons. (1) The protein may be photo bleaching. Increase the concentration of protein to obtain sufficient signal throughout the titration and adjust for photobleaching in analysis. (2) The protein may be aggregating. Adjust conditions to reduce aggregation. (3) The protein may be sticking to the pipette tip, or to the cuvette. To assess if it is sticking to the cuvette collect several spectra at different concentrations and check for linear dependence of signal. If protein is sticking to the pipette obtain low- retention tips. Pipette tips can also be treated in the lab using a siliconizing agent
An increase in fluorescence is detected in the buffer reference titration	The buffer is likely contaminated. Fresh buffer should be used for all fluorescence experiments
An increase in fluorescence is detected in the peptide reference titration	Confirm that the substrate is not intrinsically fluorescent. If not, the peptide stock may be contaminated