Liquid handling

The use of liquid handling is essential for the automation of the system. We suggest that special caution be used when selecting the speed, dead volumes, and type of liquid handler to dispense the solutions. The accuracy of liquid handling systems can significantly impact the quality of the resulting data. The most important factors to keep in mind are 1) avoiding the formation of bubbles and 2) errors in sample preparation.

1. Avoid the formation of bubbles: The addition of detergents or too rapid pipetting can lead to the formation of air bubbles in the well. If air bubbles are within the detected area, they can easily be identified by an M-like Z-scan. The transition from the aqueous sample to the air bubble back to the liquid leads to a sudden drop in reflected light signal, just above the well bottom. As they can negatively affect the measurement result, air bubbles should be avoided or eliminated prior to measurement. It is therefore recommended to centrifuge the plate open for 30 sec at 1000 x g before loading into Dianthus, and air bubbles are usually efficiently removed.

The figures above illustrate well scans with an air bubble (dark blue line) in comparison to a normal well (purple line). The top figure shows the Z well scans, and the bottom shows an example of X or Y scans. Note that air bubbles are not always in the center of the well like in this example, but rather near the edge of the well. When near the edge of the well, bubbles can be identified in the X- or Y-Scan (figure at the bottom).

2. Errors in sample preparation: This causes failed well scans and TRIC traces that differ significantly from the standard.

2a) Failed well scans or aberrations in their shape can be produced by target or ligand aggregation, dust inside or outside the wells or in the bottom of the plate, scratches in the plate bottom, or if the interaction didn’t reach equilibrium. We recommend centrifuging the target at maximum speed of a benchtop centrifuge for 10 minutes at 4 °C every time that there is a new labeled protein, or after thawing an aliquot of a labeled target. Also, always keep the plates stock away from dust, blow air on the bottom of the plate to eliminate dust and use a lint free tissue to clean the bottom of the plate before inserting it in the instrument.

The figures above show well scans with different quality issues compared for a good well scan.

2b) TRIC is very dependent on mixing and aggregation. Poor mixing results in a rapid increase in relative fluorescence after switching on the IR laser, so that it even exceeds the initial fluorescence. The convective flow during heating leads to mixing of the inhomogeneous sample. Initially there are fewer fluorescent molecules in the focus than after mixing, so that a too low value for the initial fluorescence is assumed. In addition, X- and Y- well scan anomalies can occur due to the inhomogeneous distribution of the fluorescence, e.g. an M-like shape. If a measurement shows the described characteristics, it is advisable to thoroughly mix the wells again or to check the mixing properties of the liquid handling system used. It is often sufficient to increase the number of mixing cycles, or the mixing volume used. We recommend at least 15 aspiration and dispense steps with 80% of the total volume or alternatively the use of efficient plate shakers.