Random Changes in Initial Fluorescence

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 the changes in initial fluorescence observed are not random but rather 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 pre- paration 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 low data quality.
• 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 uncovered to remove them.
• Do not prepare less than a 20 µL reaction volume.