The initial fluorescence of all capillaries in a binding assay should be identical since the same amount of fluorescent molecule is added to each sample. However, two types of fluorescence changes can occur:

• Random fluorescence changes
• Ligand-dependent fluorescence changes

If you have observed changes in the initial fluorescence that exceed a threshold of +/- 20% from the average you can follow the instructions below. However, in case you are using the MO.Control software, random fluorescence changes are detected automatically, while the software provides guidance and recommendation how to proceed with assay optimization. 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 this article 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 micro reaction tubes) or partial unfolding.
• Poor mixing and/or pipetting:
• Make sure that all pipettes are working properly and recently calibrated.
• Mix samples thoroughly by pipetting several times up and down with at least half of the total volume. Do not vortex or snip/flick the sample.
• Ensure that no liquid is lost inside and outside of the tip.
• Avoid air bubbles.
• Do not prepare less than a 10 µl ligand + 10 µl fluorescent molecule reaction.