Purified NT5B protein in 25 mm HEPES, 150 mm NaCl, pH 7.4, was fluorescently labeled using the MonolithTM NT protein labeling kit RED-NHS (Amine Reactive) dye, following the manufacturers' instructions (NanoTemper Technologies). All stock protein samples were centrifuged for 10 min prior to setting up the experiment. The labeled NT5B protein was diluted to 2.5 nm final concentration, in the appropriate buffer for each experiment (25 mm HEPES, 150 mm NaCl, 0.05% Tween 20, with either 5 mm CaCl2 or 5 mm EGTA, at pH 7.4 or 6). The nonlabeled recombinant CT5B protein was serially diluted 1:1 in the same buffer (starting concentration of 6 μm ) and an equal volume of the labeled protein was mixed with each dilution. The samples were loaded onto MonolithTM NT.115 Standard Treated Capillaries (NanoTemper Technologies) and thermophoresis was measured using a MonolithTM NT.115PICO instrument (NanoTemper Technologies) with NT Control software version 1.0.1 at room temperature with 5 s/30 s/5 s laser off/on/off times, respectively, and 15% LED power and 40% IR-laser (MST) power. Experiments were carried out in triplicate and data from three independently pipetted measurements were analyzed using NT Affinity Analysis software version 2.0.2, using the KD model of fit.
Monolith nt 115 standard treated capillaries
Manufactured by NanoTemper
Sourced in Germany
The Monolith NT.115 standard-treated capillaries are lab equipment designed for use with the Monolith instrument. They are engineered to enable sample analysis within the instrument.
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8 protocols using monolith nt 115 standard treated capillaries
1
Fluorescent Labeling and Thermophoresis Assay for Protein-Protein Interactions
2
Fluorescent Probe Binding Kinetics
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Stability constants of the complex between the primer or Flu-dUTP (fluorescein-5(6)-carboxaminocaproyl-[5-{3-aminoallyl}-2′-deoxyuridine-5′-triphosphate]) and the enzyme were determined by means of a Monolith NT.115 system (NanoTemper Technologies) using standard capillaries (MonolithTM NT.115 Standard Treated Capillaries). Each point on the titration curves was determined by measuring fluorescence intensities of individual solutions (10 μl) containing a primer or Flu-dUTP (0.5 μM) and the enzyme (0.001–12.500 μM) in a buffer (50 mM Tris–HCl, pH 8.0, 9% of glycerol, and 5 mM MgCl2 or 1 mM MnCl2 or CoCl2) at 37°C. To calculate the dissociation constants, the experimental data were processed in the DynaFit software (BioKin) in accordance with the single-stage binding model (Fig 12 ).
3
Measuring CPIII Binding Affinities
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The affinities of CPIII for ABCB6-∆TMD0 and other mutant proteins were measured by MST, based on the changes in initial fluorescence, as described previously (Krossa et al., 2018 (link)). Briefly, 5 µM of fluorophore CPIII was added to serial 2-fold dilutions of each protein from 66.6 µM to 2.0 nM. The mixtures were incubated for 20 min at room temperature and loaded onto Monolith NT.115 standard-treated capillaries (NanoTemper Technologies, Germany). Changes of initial fluorescence intensity were monitored with a Monolith NT.115 pico device, and plotted against protein concentration to calculate Kd values, and the results were analyzed with MO.Affinity Analysis software.
4
Microscale Thermophoresis of NTCP-GFP
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Recombinant NTCP-GFP and GFP proteins were incubated with different concentrations of compounds in 50 mM potassium phosphate buffer (pH 7.0) containing 100 mM NaCl, 0.2% BSA, and 0.005% DDM (n-Dodecyl-β-D-maltopyranoside) for one hour at room temperature. Samples were loaded on Monolith NT.115 Standard Treated Capillaries (Nano Temper Technologies) and analyzed with a Monolith NT.115 Blue Red microscale thermophoresis instrument.
5
Quantifying Protein-DNA Interactions using MST
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MST experiments were performed on a Monolith NT.115 instrument using a blue filter (NanoTemper Technologies) and Alexa 488–labeled DNA for non–α-satellite DNA (488-forward: 5′-ATTCCATGGCACCGTCAAGGCTGAGAACGGGAAGCTTGTC-3′; reverse: 5′-GACAAGCTTCCCGTTCTCAGCCTTGACGGTGCCATGGAAT-3′) and a centromeric human α-satellite DNA consensus (Vissel and Choo, 1987 (link); 488-forward: 5′-ATTCAACTCACAGAGTTGAACCTTCCTTTTCATAGAGCAG-3′; reverse: 5′-CTGCTCTATGAAAAGGAAGGTTCAACTCTGTGAGTTGAAT-3′). The α-satellite or non–α-satellite DNA duplexes were annealed in MST buffer (100 mM NaCl, 20 mM Tris-HCl, pH 7.4, and 1 mM DTT). For DNA-binding assays, a dilution series of proteins from 200 to 0.1 µM and 0.04 µM annealed α-satellite or non–α-satellite DNA duplexes were incubated in MST buffer for 2 h in ice before being loaded into Monolith NT.115 standard treated capillaries (NanoTemper Technologies). Measurements were performed with power settings of 80% for MST and 50% for the light-emitting diode. Data analysis and Kd calculation were performed using the NanoTemper analysis software.
6
Determining CrtJ-AerR Binding Affinity
7
Microscale Thermophoresis for Peptide-Protein Binding
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Binding of 9-mer peptide (RRYQNSTCCy-5L) to DM-purified ΔTMD0 was monitored by microscale thermophoresis as described previously40 (link). Briefly, 9-mer peptide was incubated with a 10x molar excess of Cy5 dye (Thermo Fisher Scientific) in 50 mM HEPES–NaOH pH 7.5 at RT for 2 h. The labeled peptide was purified from the excess dye with a desalting column. Cy5-labeled peptide (0.2 μM) was added to serial 2-fold dilutions of protein. The mixtures were incubated for 10 min at RT and loaded onto Monolith NT.115 standard-treated capillaries (NanoTemper Technologies). Thermophoretic movements were induced by infrared laser activation. Subsequent fluorescence changes were monitored with a Monolith NT.115 pico device and plotted against protein concentration. The results were analyzed with MO.Affinity Analysis software.
8
EGFP-Fused Protein Binding Assay
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MST measurements were performed using the Monolith NT.115 instrument and the Monolith NT.115 standard treated capillaries (NanoTemper Technologies) according to the manufacturer's instructions. The capillaries were loaded with a mixture of a recombinant EGFP-fused protein at a constant concentration of 100 nM in the SEC buffer and its binding partner in the indicated series of concentrations. The changes in thermophoresis were analyzed with the Monolith NT Analysis Software.
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