Mst buffer

Microscale thermophoresis (MST) experiments were performed on a Monolith NT.115 instrument (NanoTemper Technologies). Purified His-tagged SR-B1 protein (His₆.hSR-B1(amino acids 33–444)) was labeled using the Monolith His-Tag Labeling Kit Red-tris-NTA (NanoTemper Technologies) and diluted in MST buffer (NanoTemper Technologies) to a concentration of 50 nM and was mixed with APOA1 or APOA1-Milano in MST buffer (PBS with 0.05%Tween) at indicated concentrations ranging from 1 nM to 8 μM at room temperature. Fluorescence was determined in a thermal gradient at 40% LED power and high MST power generated by a Monolith NT.115 from NanoTemper Technologies. Data were analyzed using Affinity Analysis v2.3.

The PHB-fluorescence proteins were adjusted to 8 μM with MST buffer supplemented with 0.05% Tween 20 (NanoTemper Technologies). PDD005 was dissolved in MST buffer supplemented with 0.05% Tween 20 and a series of 16 1:1 dilutions (ligand concentrations ranging from 200 µM to 6 μM) were prepared using the same buffer. For the measurement, each ligand dilution was mixed with one volume of PHB-fluorescence proteins (1 and 2 isoforms), leading to a final concentration of PHB-fluorescence proteins of 4 μM and final ligand concentrations ranging from 100 µM to 3 μM. After 10 min incubation followed by centrifugation at 10 000 × g for 10 min, the samples were loaded into standard Monolith NT.115 Capillaries and MST was measured using a Monolith NT.115 instrument (NanoTemper Technologies) at a temperature of 22 °C as described previously^{44 (link)}. We adjusted the instrument parameters to 100% LED power and medium MST power (40%). We analyzed data from two independently measuremnts (MO.Affinity Analysis software version 2.3, NanoTemper Technologies) using the signal from an MST-on time of 20 s.