Red nhs protein labeling kit

The purified recombinant proteins were dialysed into 1× PBS, and then labeled with the red fluorophore according to the protocol of Protein Labeling Kit RED-NHS (Nanotemper, Cat# L001). All the tested stock compounds (25 mM) were serially diluted into the same buffer (20 mM HEPES, pH 7.5, 150 mM NaCl) with the same final DMSO concentration (2.5%) for the MST assay. The MST experiment was performed using Monolith NT.115 instrument (NanoTemper Technologies). Labeled proteins (500 nM) were mixed with the indicated concentrations of candidate compounds in reaction buffer containing 20 mM HEPES, pH 7.4, 150 mM NaCl. The MST data were then collected under 40% infrared laser power and 20% light-emitting diode power. The data were analyzed by Nanotemper analysis software (v.1.5.41).

The equilibrium dissociation constant (K_D) values for protein–protein and protein–peptide interaction were measured using the Monolith NT.115 Capillaries (NanoTemper Technologies, München, Germany). The purified PAPP-A was firstly adjusted to a concentration of 2 μM and then fluorescently labeled by Protein Labeling Kit RED-NHS (NanoTemper Technologies, Germany) according to the manufacturer’s protocol. Peptides, substrates, and modulator proteins were serially diluted in binding buffer (PBS buffer containing 0.05% Tween-20) and then mixed and incubated with an equal volume of 24 nM labeled PAPP-A at room temperature for 30 min. The sample was loaded into the NanoTemper glass capillaries and micro-thermophoresis was carried out using 20% light excitation power and medium MST power. The data were obtained from triplicate independent experiments and the K_D values were calculated using the signal from an MST-on time of 1.5 s by the NanoTemper Monolith affinity software using 1:1 binding mode. Flag peptide (DYKDDDDK) was used as a negative control without any signals (data not shown).

The HIV-1 proteins were fluorescently labeled using Protein Labeling Kit Red-NHS (Nanotemper Technologies, München, Germany). The labeling reaction was performed according to the manufacturer’s instructions. The 20 µM protein was mixed with the dye (1:3 molar) and reacted for 30 min. The unreacted dye was removed with supplied dye removal column. The labeled proteins and tubules were diluted 20×, and 5% DMSO was added. The labeled RNA and DNA oligomers were diluted to a concentration 20 nM. Fullerene 1 was dissolved in the same buffer and a series of 16 1:1 dilutions. For the measurement, each ligand dilution was mixed with one volume of labeled molecules, which led to a final concentration of fullerene ranging from 50 µM to 15 nM. The samples were loaded into Monolith NT.115 Capillaries (NanoTemper Technologies, München, Germany). MST was measured using a Monolith NT.115 instrument (NanoTemper Technologies, München, Germany) at temperature of 25 °C. Instrument parameters were adjusted to 100% LED power and medium MST power. Data of three independent measurements were analyzed using Affinity Analysis software version 2.3 (NanoTemper Technologies, München, Germany) using the signal from an MST over 1.5 s and the initial fluorescence data.

MST experiments were performed on a Monolith NT.115 instrument (NanoTemper Technologies). Proteins were labelled with the red fluorescent dye NT-647 using the Protein Labeling Kit RED-NHS (NanoTemper Technologies). The concentration of the labelled protein was kept constant at 100 nM, while the concentration of the compound was varied. A 15-step twofold dilution series beginning at 500, 250 or 125 µM finally yielded 16 different concentrations of the tested compound [(1)–(4)]. Experiments were carried out in 10 mM HEPES buffer pH 8 containing 100 mM NaCl, 1 mM DTT, 0.05%(w/v) Tween 20, 0.25 mM MgCl₂ and 0.25 mM MnCl₂. The final samples were adjusted to 5% DMSO to ensure the solubility of the compounds. The samples were centrifuged for 5 min at 13 000 rev min⁻¹ to remove potential aggregates and the supernatant was loaded into standard treated MST-grade glass capillaries (NanoTemper Technologies). After a 5 min incubation period the MST was measured with 80% LED power and 80% infra-red laser power. K_d values were determined using the NanoTemper analysis software.