Red tris nta dye

To determine the K_d (dissociation equilibrium constant) of RbcL-DNAK2 or RbcL-DnaJ Sll1384 complexes, microscale thermophoresis (MST) was performed. For that, His-tagged proteins, namely DnaK2 or DnaJ Sll1384, were labeled with RED-tris-NTA dye (Nano Temper), following the supplier-suggested protocol. For that, 200 nM protein was incubated with 5 mM dye in 200 μl PBS-T buffer for 30 min a room temperature. For MST measurement, samples containing 4 μM or 200 nM of the labeled chaperone and unfolded RbcL (concentration ranges given in the results presenting figures) were prepared in a PBS-T buffer (phosphate-buffered saline, 0.1% Tween® 20) and loaded into a set of 16 capillaries (Monolith NT.115 MO-K022 capillaries, Nano Temper Technologies GmbH, Munich, Germany). The samples were analyzed with a Monolith NT.115 pico device under MO. Control Software (Nano Temper Technologies GmbH, München, Germany). Nano-red excitation with a LED light adjusted to 60% excitation power was used, while the infrared laser was set to high. The K_d value was calculated using the software’s built-in routines.

Proteins were labeled using Red-Tris-NTA dye (NanoTemper, Munich, Germany) and resuspended in 50 μl of phosphate-buffered saline (PBS) buffer (137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 2 mM KH₂PO₄ [pH 7.4]) with 0.05% Tween 20 to obtain a 5 μM dye solution. The labeled-protein solution containing 500 nM proteins and 40 nM dye was prepared in PBS buffer with 2% dimethyl sulfoxide (DMSO) for the assay. The 12 concentrations for each compound were obtained by 2-fold serial dilutions (0.2 μM to 400 μM). A peptide control was included to discriminate binding-specific fluorescence quenching from the loss of fluorescence due to protein precipitation. Two independent experiments were carried out in premium coated capillaries (NanoTemper, Munich, Germany) using a Monolith NT.Automated instrument (NanoTemper) with the power set at high (80%), the light-emitting diode (LED) power (pico red) set at 5%, and the on-time set at 20 s. The dissociation constant (K_d) was determined using the MO.Affinity Analysis program (NanoTemper).

Quantification of NorC’s interaction with TPP⁺ was carried out using microscale thermophoresis (Nanotemper)^{56 (link)}. The protein was labeled with red Tris-NTA dye (Nanotemper) by mixing both in an equimolar proportion. The labeled protein was mixed with TPP⁺ such that the concentration of the labeled protein and TPP⁺ were 10 nM and 0.1 mM, respectively. A total of 16 twofold serial dilutions of TPP⁺ were prepared to keep the protein concentration constant at 10 nM. The experiment was carried out with Monolith™ NT.115 MST premium-coated capillaries. For NorC-ICab complex titrations against TPP⁺, 10 nM of NorC was mixed with ICab in a 1:3 molar ratio and the starting concentration of TPP⁺ was kept at 0.1 mM. For ICab titrations against NorC, 10 nM of NorC was used with a starting concentration of 5 µM for the ICab. The curves were fitted with the single-site binding model. To assess the same with NorB and ICab, identical concentrations were taken, with NorB in place of NorC.

The binding affinity of spike to Sb23 was measured by microscale thermophoresis using the MST NT.115 device (NanoTemper Technologies). Sb23 was labeled via the His₆-tag with RED-tris-NTA dye (NanoTemper Technologies) in PBS-T (10 mM phosphate, 2.7 mM KCl and 137 M NaCl, pH 7.4 supplemented with 0.05% (v/v) Tween-20), following the manufacturer’s instructions. A 2-fold dilution series of spike protein was prepared in PBS-T with the highest concentration in the assay being 1250 nM. A 50 nM final concentration of labeled Sb23 was mixed with the substrate dilution series and incubated for 10 min at room temperature before loading the samples in standard capillaries. For the competition assay, the same procedure was used but the spike dilution series were prepared in PBS-T buffer containing 800 nM of ACE2 (200 nM final concentration in the assay) and incubation of 10 min prior to mixing with labeled Sb23. All measurements were performed in triplicate using 35% LED and medium MST power. The data were analyzed using the MO. Affinity Analysis software (NanoTemper Technologies). Figures were prepared using Prism 8 (GraphPad).

SAM binding to purified native and mutated MtbCbs was evaluated using microscale thermophoresis experiment (Nanotemper Technologies, Munich, Germany), that measures binding affinity of SAM. For this assay, the final protein concentration used was 100 nM. The protein was labeled with 100 nM red tris-NTA dye (Nanotemper Technologies, Munich, Germany) in the C terminus of the His-tag. Monolith NT.115 capillaries (Nanotemper Technologies, Munich, Germany) were used in each experiment. In the binding assay, the protein concentration for both native and mutated was kept constant. Both proteins were incubated with 16 twofold serial dilutions of the ligand SAM. The ligand was solubilized in a protein-containing buffer [50 mM Hepes (pH 7.4), 150 mM NaCl, and 3% glycerol]. The starting concentration for the ligand SAM was 500 μM for all the cases.