Glass capillaries

MST was carried out using a Monolith NT.115 instrument (NanoTemper). Experiments were performed at 22°C in 25 mM MES (pH 6.5) and 100 mM NaCl. Both PepT1^ECD and PepT2^ECD were mutated to change a surface-exposed serine to a cysteine (PepT1^ECD-S437C and PepT2^ECD-S427C) and labeled with the blue maleimide labeling kit MO-L006 (NanoTemper). A range of concentrations of the required ligand (range 0.03–1,000 μM) was incubated with 1.5  μM of purified labeled protein. The sample was loaded into the NanoTemper glass capillaries and microthermophoresis was carried out using 100% LED power and 80% MST. K_Ds were calculated using the mass action equation via the NanoTemper software from duplicate reads of triplicate experiments.

The thermophoresis experiments with fluorescently labeled monomeric and oligomeric $\alpha$ -synuclein were performed as described previously (Wolff et al., 2016 (link)), using a Monolith instrument (Nanotemper, Munich, Germany) and glass capillaries (Nanotemper, Munich, Germany) with hydrophobic coating (oligomeric $\alpha$ -synuclein) or uncoated (monomeric $\alpha$ -synuclein). A two-fold dilution series of AS69 in 20 mM phosphate buffer pH 7.4 with 50 mM NaCl was prepared and then either 10 μl of 5x diluted oligomers (corresponding to 0.6–1.2 μM) or 1 μM labelled monomer was added to each sample of the dilution series. We performed the binding experiments under these buffer conditions for optimal comparability with previous ITC experiments of AS69 binding to monomeric $\alpha$ -synuclein (Mirecka et al., 2014 (link)).
MST experiments were performed at 40% laser power and 75% LED power (oligomers) or 60% laser power and 20% LED power (monomers). For calculation of the relative change in fluorescence from thermophoresis, the cursors were set before the temperature jump followed by 5 s after the temperature jump (oligomers) and 45 s after the temperature jump (monomers).