Monolith nt labelfree instrument

Manufactured by NanoTemper

Sourced in Germany

The Monolith NT.LabelFree instrument is a lab equipment product from NanoTemper. It is designed to measure molecular interactions without the need for labels. The core function of the Monolith NT.LabelFree is to provide quantitative data on binding kinetics and affinities between molecules.

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Lab products found in correlation

Nt labelfree standard treated capillaries, by NanoTemper (3 mentions) Monolith nt standard treated capillaries, by NanoTemper (3 mentions) Premium coated capillaries, by NanoTemper (2 mentions) Nt analysis software version 1, by NanoTemper (2 mentions) Cacl2, by Carl Roth (1 mentions) Mgcl2, by Carl Roth (1 mentions) Pluronic f 127, by Merck Group (1 mentions) Analysis software version 1, by NanoTemper (1 mentions) Standard grade capillaries, by NanoTemper (1 mentions) Monolith nt protein labeling kit red nhs, by NanoTemper (1 mentions) Monolith nt 115, by NanoTemper (1 mentions) Prism version 6, by GraphPad (1 mentions) Amicon spin column, by Merck Group (1 mentions) Chelex, by Bio-Rad (1 mentions)

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Monolith NT.LabelFree (15 citations) Monolith instrument (4 citations)

20 protocols using monolith nt labelfree instrument

Integrin αIIbβ3 Binding Kinetics Analysis

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Since integrin α_IIbβ₃ is a membrane protein, we used NT. LabelFree to prevent the label from affecting the results. Titration series with ligand concentrations varying between 0 and 1,000 mM were prepared in the Prometheus NT.48 optimized buffer (Figure S1). SD-test (the automatic pre-detection of MST to detect the stability of protein signal) was required before measurement.
Approximately, 3 µl was loaded into NT.LabelFree standard-treated capillaries (Nanotemper). MST experiments were performed at 40% MST (infra-red laser) power and 60% LED power at 25°C using the Monolith NT.LabelFree Instrument (Nanotemper). Ratios between normalized initial fluorescence and after temperature-jump and thermophoresis were calculated and averaged from five to nine independent runs. Means of fluorescence intensity obtained by the MST measurements were fitted, and the resultant Kd values were given together with an error estimation from the fit by the built-in formula of the analysis software.

Yang Y.F., Wu S.T., Liu B., Xie Z.T., Xiong W.C., Hao P.F., Xiao W.P., Sun Y., Ai Z.Z., You P.T, & Wu H.Z. (2019). A Novel Antiplatelet Aggregation Target of Justicidin B Obtained From Rostellularia Procumbens (L.) Nees. Frontiers in Pharmacology, 10, 688.

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GLUT Protein Ligand Binding Assay

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Purified GLUT3 proteins, WT or mutants, were diluted in the buffer containing 25 mM MES 6.0, 150 mM NaCl, and 0.06% (w/v) CYMAL-6. Purified GLUT1 was diluted with the buffer containing 25 mM MES 6.0, 150 mM NaCl, and 0.02% (w/v) DDM. Inhibitors with serial dilutions were mixed with indicated protein. The mixture was loaded to MO-Z002 capillaries at room temperature. MST analyses were conducted on a Monolith NT. Label-free instrument (NanoTemper Technologies GmbH) with 20% LED power and 60% MST power. The MST data was analyzed by Monolith NT.115.

Wang N., Zhang S., Yuan Y., Xu H., Defossa E., Matter H., Besenius M., Derdau V., Dreyer M., Halland N., He K.H., Petry S., Podeschwa M., Tennagels N., Jiang X, & Yan N. (2022). Molecular basis for inhibiting human glucose transporters by exofacial inhibitors. Nature Communications, 13, 2632.

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Binding Kinetics of Mirolysin and Calcium

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A Monolith NT.LabelFree instrument (NanoTemper Technologies GmbH, Munich, Germany) was used to analyze the binding interactions between recombinant mirolysin and calcium (Ca²⁺). Calcium at concentrations ranging from 6.1 to 10 000 μm was incubated with 375 nm mirolysin (Mircat^E225A) in assay buffer (5 mm Tris, 50 mm NaCl, 0.02% NaN₃, 0.05% Pluronic F-127, pH 7.5) for 5 min at 20°C. The samples were loaded into Monolith NT.LabelFree Standard Treated glass capillaries, and an initial fluorescence measurement was taken followed by thermophoresis measurements using 20% of power of a light-emitting diode (LED) and 20% MST power, respectively. K_d values were calculated using MO.Affinity Analysis software. The experiment was performed in triplicate.

Koneru L., Ksiazek M., Waligorska I., Straczek A., Lukasik M., Madej M., Thøgersen I.B., Enghild J.J, & Potempa J. (2017). Mirolysin, a LysargiNase from Tannerella forsythia, proteolytically inactivates the human cathelicidin, LL-37. Biological chemistry, 398(3), 395-409.

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Calcium and Magnesium Binding to SNAP25

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Titration series with 10 μM SNAP25 and CaCl₂ or MgCl₂ (Carl Roth) concentrations varying between 0 and 1000 mM were prepared in TBS containing 0.1% [v/v] Pluronic F-127 (Sigma, St. Louis, MO). Approximately 3 μl were loaded into NT.LabelFree Standard treated capillaries (Nanotemper). MST experiments were performed at 40% MST (infra-red laser) power and 30% LED power at 37 °C using the Monolith NT.LabelFree Instrument (Nanotemper). Ratios between normalized initial fluorescence and after temperature-jump and thermophoresis were calculated and averaged from five to nine independent runs (half of the runs were incubated for 30 minutes at room temperature (RT) before the measurement while the other half were directly measured). In few cases MST traces showed signs of protein precipitation, or large deviations from the average initial fluorescence, and were not included in the analyses.

Batoulis H., Schmidt T.H., Weber P., Schloetel J.G., Kandt C, & Lang T. (2016). Concentration Dependent Ion-Protein Interaction Patterns Underlying Protein Oligomerization Behaviours. Scientific Reports, 6, 24131.

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Microscale Thermophoresis Analysis of Clitocine Binding to 80S Ribosomes

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Binding of clitocine to HeLa 80S ribosome was determined using microscale thermophoresis (MST) (Beckert et al. 2015 (link)). Purified 80S ribosomes (Khatter et al. 2014 (link)) were diluted to a final concentration of 20 pM in 1× MST buffer (NanoTemper Technologies). Clitocine or G418 was titrated with concentration ranges of 50–0.003 µM and 10 mM to 0.3 µ, respectively. The 80S ribosomes were then incubated at 25°C for 20 min and 10 µL from each reaction was loaded into premium coated capillaries (NanoTemper Technologies). The thermophoretic response was then measured by monitoring the change in tryptophan fluorescence from the ribosomes with a Monolith NT label free instrument (NanoTemper Technologies) at 20% and 10% infrared (IR) laser power. Change in fluorescence due to binding upon IR laser-mediated heating was measured and plotted against clitocine or G418 concentration. Binding constants for G418 were calculated as averages from three independent experiments.

Friesen W.J., Trotta C.R., Tomizawa Y., Zhuo J., Johnson B., Sierra J., Roy B., Weetall M., Hedrick J., Sheedy J., Takasugi J., Moon Y.C., Babu S., Baiazitov R., Leszyk J.D., Davis T.W., Colacino J.M., Peltz S.W, & Welch E.M. (2017). The nucleoside analog clitocine is a potent and efficacious readthrough agent. RNA, 23(4), 567-577.

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Ery binding affinity to KpAcrB

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The MST experiment was performed using a Monolith NT.Labelfree instrument (NanoTemper Technologies GmbH, Munich, Germany) coupled with premium standard capillaries (NanoTemper Technologies, Munich, Germany). A 16-point dilution series of Ery was made by dissolving Ery in 20 mM Na-HEPES (pH 7.5) and 0.02% DDM. Each Ery solution was then mixed with the KpAcrB protein solution in 20 mM Na-HEPES (pH 7.5) and 0.02% DDM. After 15-min incubation, these samples were filled into 16 different premium standard capillaries, respectively. The final concentration of KpAcrB in each capillary was 500 nM. The final concentration of Ery ranged between 15 nM and 500 μM. Measurements were taken with excitation wavelength at 280 nm and emission wavelength at 360 nm. For each measurement, MST signal was recorded within 25 s, and laser-on time was set at 20 s and laser-off time was set at 5 s. The experiment was repeated three times to ensure reproducibility.

Zhang Z., Morgan C.E., Bonomo R.A, & Yu E.W. (2023). Cryo-EM Structures of the Klebsiella pneumoniae AcrB Multidrug Efflux Pump. mBio, 14(3), e00659-23.

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Binding Affinities of Sialic Acids to SaSiaT

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The binding affinities for two sialic acids and purified SaSiaT were determined using microscale thermophoresis. Experiments were performed on a Monolith NT.LabelFree instrument (NanoTemper Technologies) (Wienken et al., 2010 (link); Soares da Costa et al., 2016 (link); Stifter et al., 2018 (link)). Purified SaSiaT was diluted to 2 μM in PBS buffer supplemented with 0.0174% (w/v) DDM, and incubated with Neu5Ac (from 0.3 μM to 10 mM), and Neu5Gc (0.08 μM to 2.5 mM), for 5 min prior to taking measurements. The samples were loaded into Monolith NT Standard Treated Capillaries (NanoTemper Technologies). Microscale thermophoresis measurements were carried out at 25°C using 20% LED power, and 20% microscale thermophoresis infrared laser power. The dissociation constants (K_d) were determined using the mass action equation via the NT Analysis software version 1.5.41 (NanoTemper Technologies), using the signal from Thermophoresis + T-jump for triplicate experiments.

North R.A., Wahlgren W.Y., Remus D.M., Scalise M., Kessans S.A., Dunevall E., Claesson E., Soares da Costa T.P., Perugini M.A., Ramaswamy S., Allison J.R., Indiveri C., Friemann R, & Dobson R.C. (2018). The Sodium Sialic Acid Symporter From Staphylococcus aureus Has Altered Substrate Specificity. Frontiers in Chemistry, 6, 233.

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Microscale Thermophoresis for Affinity Measurements

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Affinity measurements using microscale thermophoresis (MST) were carried out with a Monolith NT. LabelFree instrument (NanoTemper Technologies)19 42 (link). 2,6-PDC diluted in water (5.0 mM to 2.4 μM) was mixed 1:1 with the enzyme pre-incubated with 150 μM NAD⁺ or NADP⁺, yielding a final DHDPR concentration of 10 μM and a dilution series of 2.5 mM to 1.2 μM of 2,6-PDC. Controls were performed in the absence of NAD⁺/NADP⁺, with water added instead, but with the same dilution series for 2,6-PDC. All experiments were incubated for 30 min at 30 °C, before applying samples to Monolith NT Standard Treated Capillaries (NanoTemper Technologies). Thermophoresis was measured at 30 °C with 5 s/30 s/5 s laser off/on/off times. Experiments were conducted at 30% LED power and 40% MST IR-laser power. Data from three independently performed experiments were fitted to the single binding site model (NT. Analysis software version 1.5.41, NanoTemper Technologies) using the signal from Thermophoresis + T-Jump.

Christensen J.B., Soares da Costa T.P., Faou P., Pearce F.G., Panjikar S, & Perugini M.A. (2016). Structure and Function of Cyanobacterial DHDPS and DHDPR. Scientific Reports, 6, 37111.

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Quantifying Akt-Inhibitor Binding Kinetics

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Using 0.1 M Tris-HCl buffer (pH 7.2) containing 0.05% Tween-20, a 1:1 serial dilution was prepared for G7 to yield 16 working solutions, with the highest concentration being 200 µM. Each of these G7 solutions was mixed with 1 µM of purified Akt sample prepared in the same 0.05% Tween-20 Tris-HCl buffer. The Akt samples tested include recombinant Akt PH domain, inactive Akt, and active Akt obtained from Millipore. After 15-min incubation the samples were centrifuged at 14,000 g at 4 °C before loading into premium coated capillaries (NanoTemper Tech.) for MST measurements. The MST experiments was conducted on a Monolith NT.LabelFree instrument (NanoTemper Tech.), at 20% LED power and 40% MST power. Data analyses were performed using the NanoTemper analysis software.

Huang B.X., Newcomer K., Kevala K., Barnaeva E., Zheng W., Hu X., Patnaik S., Southall N., Marugan J., Ferrer M, & Kim H.Y. (2017). Identification of 4-phenylquinolin-2(1H)-one as a specific allosteric inhibitor of Akt. Scientific Reports, 7, 11673.

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Microscope Thermophoresis of Compound Interactions

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Microscope Thermophoresis experiments were performed using 1:1 mixture of PAC and compounds in standard grade capillaries (Nanotemper Technologies). Samples were incubated at 25 °C within the capillaries for 5 mins prior to measurement. All measurements were conducted using Monolith NT.LabelFree instrument (NanoTemper Technologies) at 25 °C. Assays were conducted at 20% IR-laser power and Medium MST powers. The buffer used for the experiment was 20 mM Tris, 200 mM NaCl and 15% Glycerol, pH 8. Stock solutions of test compounds were first diluted in buffer to their respective initial concentrations. A two-fold dilution series of the compounds were then prepared for MST measurement.

Lo C.Y., Li O.T., Tang W.P., Hu C., Wang G.X., Ngo J.C., Wan D.C., Poon L.L, & Shaw P.C. (2018). Identification of influenza polymerase inhibitors targeting C-terminal domain of PA through surface plasmon resonance screening. Scientific Reports, 8, 2280.

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