Technical Notes

7 TECHNICAL NOTE ©2019 NanoTemper Technologies, Inc. South San Francisco, CA, USA. All Rights Reserved. one for the positive control, were prepared as a 10-fold concentrated stock solution. These stock solutions contained 50 nM labelled G9a (10x reference stock) or 50 nM labelled G9a mixed with 1 mM ligand SAM (10x positive control stock), respectively. Next, 18 μL of each buffer solution from the 96-deep-well plate of the Buffer Exploration Kit were transferred into 4 wells of a Dianthus 384-well plate (Cat# DI-P001A, NanoTemper Technologies). This step was executed using an 8-channel Precision XS system for liquid handling (BIOTEK). For each buffer, the four wells were arranged next to each other forming a square as shown in figure 2, le panel. Next, 2 µL of the 10x reference stock was added to every well in odd-numbered columns of the prefilled 384-well plate and mixed thoroughly by pipetting up and down. The wells on even-numbered columns were filled with 2 µL of the 10x positive control stock solution and mixed thoroughly by pipetting up and down. For these liquid handling steps, a STARlet system (HAMILTON) was used. The final concentration in the 384-well plate was 5 nM for G9a and 100 µM for S-adenosyl-L-methionine (SAM), respectively. A er both stock solutions were added, the 384-well microplate was equilibrated for 2.5 hrs at room temperature in the dark and then centrifuged for 30 sec at 1000 x g before loading into a Dianthus NT.23PicoDuo for data acquisition. The temperature of the system was set to 25°C and samples were measured with 5 sec on-time for the IR-laser. Both optical systems in Dianthus were used in parallel, resulting in an overall measurement time of ~30 min for 384 wells. Measured fluorescence values collected are displayed as relative fluorescence as standard. The Signal Quality (SQ), which is used to evaluate the buffer screening, is defined as the ratio between Signal Area and Noise Area. Signal Areas refer to areas that lie between a reference trace and a positive control trace. Noise Areas are those areas between two references (Reference Area) or two positive controls (Ligand Area). By this definition, the value of the SQ is reduced when the reproducibility of the duplicates deteriorates and when the difference between reference curves and positive control curves decreases. A high SQ value is therefore indicative of a high-quality measurement signal. The SQ values obtained from each condition of the buffer screen are depicted in a 96-sectioned heatmap. Data were analyzed with NanoTemper's DI.Control so ware v1.0.1.

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