Monolith nt 115 instrument

Daidzein was diluted with MST buffer (50 mM HEPES, 150 mM NaCl, 10 mM MgCl2, 0.05% Tween-20, pH 7.0) to 1 mM. Lipoxygenase from soybean (L7395, Sigma, China) was labeled by a Monolith NT^TM RED-NHS Second Generation Protein Labeling Kit based on the manual (NanoTemper Technologies GmbH, Munich Germany) and diluted with MST buffer to 100 nM. The mixture of daidzein and labeled-ALOX15 was incubated at room temperature for 30 min and their binding affinity was analyzed by microscale thermophoresis (NanoTemper Monolith Instrument NT.115). Red-laser was absorbed by mixed aqueous solutions of different concentrations in 12 capillaries. Fluorescence was determined in a thermal gradient at 40% MST power with laser off/on times of 5–20 s, and data was analyzed by MO Affinity Analysis v2.3.

Microscale thermophoresis (MST) was performed to qualitatively analyze the interaction between BpfD-In and CRP. Proteins were heterologously expressed and purified. His₆-CRP was dialyzed with PBS-glycerol buffer. GST and GST-BpfD-In were freeze-dried after dialysis with deionized water. Subsequently, 100 μl of 10 μM His₆-CRP was labeled with NHS NT-647 dye using the Red-NHS 2nd Generation Labeling Kit (NanoTemper, Germany), which was eluted with a reaction buffer (PBS-glycerol buffer: Pull-down buffer = 1:1). Next, 10 μl of labeled His₆-CRP was mixed with 10 μl of 5 μM GST or GST-BpfD-In or with the same volume of reaction buffer, respectively. Finally, samples were loaded into capillaries and analyzed using a Monolith Instrument NT.115 (NanoTemper, Germany) in Binding Check mode. Both the LED power and MST power were set to 20%.

Recombinant human CALR del52 was labeled with NHS- chemistry according to the manufacture’s instruction (Protein Labeling Kit RED-NHS 2^nd Generation, NanoTemper Technology), referred as the “target protein”. Briefly, the target protein (at 10 μM) was incubated with the dye solution in the labeling buffer (130 mM NaHCO₃, 50 mM NaCl, pH 8.2–8.3) for 1 h on ice. The dye carries a reactive NHS-ester group that reacts with primary amines (lysine residues) to form covalent bonds. The surplus of the dye not bound to the target protein was removed through passage on a resin column prior to elution of the target in the equilibration buffer (Tris-HCL 1 M, pH 7.6). For MST measurement, the CALR del52-NHS was used at 20 nM final concentration in MST buffer (Tris based supplemented with 0.01% of tween 20).
TpoR D1–D4 (the “ligand”) remained label free. Serial dilutions (0.15 nM to 5 μM) of the ligand (TpoR D1-D4) were performed to titer the target protein (CALR del52). The measurements were performed on a NanoTemper monolith NT.115 instrument (NanoTemper technologies, Germany) at 40% LED and medium MST-power with a standard 5 s. before, MST-on for 30 s. and 5 s. after MST-off.

Recombinant human PKM2 (ab89364, Abcam) was labeled with a fluorescent dye (Pico-RED) using Monolith™ RED-NHS Protein Labeling Kits (MO-L011). The labeling procedure and the subsequent removal of free dye were performed according to the manufacturer’s instructions. The concentration of PKM2 was kept constant at 20 nM. A serial dilution of MC-RR was prepared and mixed with PKM2 by 1:1 (v/v). The highest concentration of MC-RR was 200 μM. The mixed samples were loaded into standard treated MO NT.115 capillaries (MO-K022, NanoTemper). Binding measurements were conducted using a NanoTemper Monolith NT.115 Instrument (NanoTemper Technologies GmbH) and carried out when LED power was set to medium and laser power set to 40%. The MST data of independent measurements were processed using Prism GraphPad software to calculate the binding coefficient (Kd) for interacting partners.

Measurements were performed using a NanoTemper Monolith™ NT.115 instrument (NanoTemper Technologies GmbH, München, Germany). Protein samples were labelled with the amine reactive dye NT-647 (or NT-495) using the Monolith™ NT.115 Protein Labeling Kit RED-NHS (or Monolith™ NT.115 Protein Labeling Kit BLUE-NHS). Labelling levels (generally in the range 0.3–0.4 dye molecules per protein molecule) were determined using calculated extinction coefficients for the protein and ε647 = 250,000 M⁻¹ cm⁻¹ (or ε495 = 70,000 M⁻¹ cm⁻¹) for the dye concentration. In a typical experiment 20 ml aliquots of a 100 nM stock solution of labelled protein were mixed with 20 ml aliquots of a serial dilution of binding partner. These solutions were then loaded into standard treated capillaries and MST measurements were performed at 25 °C using 20–40% LED power and 40–60% IR-Laser power. The laser Laser-On time was 30 s and Laser-Off time 5 s. All measurements were performed at least five times.