Random changes in Initial Fluorescence are usually the result of sample inhomogeneities (such as Target Aggregation). Dianthus software recognizes random fluorescence changes and highlights them. Most often, centrifugation of Target and Ligand stock solutions, and the addition of low amounts of Detergents, can help to alleviate sample inhomogeneity. If you have observed random changes in the initial fluorescence that exceed a threshold of ± 20 % from the average, you can follow the instructions below. If however, the changes in initial fluorescence you observed are not random but rather clearly a function of the concentration of ligand added to the sample, please refer to the article on Ligand-Induced Fluorescence Change for further guidance.

Underlying mechanisms and recommendations:

• Poor sample quality: The buffer is not optimal for the protein, leading to slight aggregation, varying amount of sample (material might be lost during sample preparation due to adsorption to pipet tips and plastic labware) or partial unfolding.
• Poor mixing and/or pipetting: Make sure that all pipettes are working properly and were recently calibrated. Mix samples thoroughly by pipetting several times up and down with at least half of the total volume. Inefficient mixing is one of the most common sources of bad data quality.
• The interaction didn’t reach equilibrium: Make sure that the minimum time to reach equilibrium of the interaction is reached before measuring the plate. We recommend using at least 30 minutes incubation to make sure that the interaction has reached equilibrium. However, in some cases that the interaction has low kinetics, it is possible that an incubation of 1, 2 or 4 h might be needed to reach equilibrium. If the reaction is not in equilibrium, the wells and values might have random errors.
• Do not vortex protein samples.
• Ensure that no liquid is lost inside and outside of the pipette tip used.
• Avoid air bubbles. If they occur, centrifuge the Plates open to remove them.
• Do not prepare less than a 20 µL reaction volume.