The Mtr LpqY protein was labeled using the amine reactive RED-NHS dye (3 μM) (second generation, NanoTemper Technologies) and a constant concentration of Mtr LpqY (2.6 μM). Excess dye was removed by size exclusion chromatography (Superdex 200 10/300 column [GE Healthcare] using 50 mM HEPES, 300 mM NaCl, pH 7.5). The compounds were prepared in PBS containing 0.05% Tween 20, and the final concentration of the protein in the assay was 500 nM. The samples were loaded into the MonoLith NT.115 standard treated capillaries and incubated for 10 min before analysis using the MonoLith NT.115 instrument (NanoTemper Technologies) at 21 °C using the auto-select excitation power (20%) and medium laser power. The binding affinities were calculated using a single-site binding model using the MST NT Analysis software (version 7.0). All experiments were carried out in triplicate.
Monolith nt 115
Manufactured by NanoTemper
Sourced in Germany, United States, United Kingdom
The Monolith NT.115 is a compact, high-performance instrument designed for biomolecular interaction analysis. It utilizes the principle of Microscale Thermophoresis (MST) to detect and quantify molecular interactions in a label-free and solution-based environment. The core function of the Monolith NT.115 is to measure binding affinities, kinetics, and thermodynamics of a wide range of biomolecular interactions, including protein-protein, protein-small molecule, and protein-nucleic acid interactions.
Automatically generated - may contain errors
Lab products found in correlation
Mo affinity analysis software, by NanoTemper (34 mentions)
Nt analysis software, by NanoTemper (24 mentions)
Affinity analysis software, by NanoTemper (19 mentions)
Monolith nt protein labeling kit red nhs, by NanoTemper (19 mentions)
Standard capillaries, by NanoTemper (14 mentions)
Premium coated capillaries, by NanoTemper (13 mentions)
Monolith nt capillaries, by NanoTemper (13 mentions)
Mo affinity analysis v2, by NanoTemper (13 mentions)
Red nhs protein labeling kit, by NanoTemper (11 mentions)
Analysis software v 1, by NanoTemper (9 mentions)
Premium capillaries, by NanoTemper (9 mentions)
Monolith nt 115 premium capillaries, by NanoTemper (9 mentions)
Red tris nta, by NanoTemper (8 mentions)
Monolith nt protein labeling kit red, by NanoTemper (8 mentions)
Monolith nt 115 capillaries, by NanoTemper (8 mentions)
Monolith protein labeling kit red nhs 2nd generation, by NanoTemper (7 mentions)
Silica capillaries, by Molex (7 mentions)
Standard treated capillaries, by NanoTemper (7 mentions)
Nt647, by NanoTemper (7 mentions)
Monolith his tag labeling kit red tris nta, by NanoTemper (7 mentions)
643 protocols using monolith nt 115
1
Mtr LpqY Protein Labeling and Binding Affinity
2
CEACAM1a-MHV Spike Protein Binding Assay
3
CEACAM1a-MHV S Protein Binding Kinetics
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Solution MicroScale Thermophoresis (MST) binding studies were performed using standard protocols on a Monolith NT.115 (Nanotemper Technologies). In brief, recombinant CEACAM1a ectodomain protein was labelled using the RED-NHS (Amine Reactive) Protein Labelling Kit (Nanotemper Technologies). The MHV S ectodomain protein was serially diluted in 20 mM Tris-HCl, pH 7.5, 100 mM NaCl and the labelled recombinant CEACAM1a was added to a final concentration of 500 nM before overnight incubation at 4 °C. The CEACAM1a concentration was chosen such that the observed fluorescence was approximately 1,000 U at 40% LED power. The samples were loaded into standard-treated Monolith capillaries and were measured by standard protocols using a Monolith NT.115, NanoTemper. The changes in the fluorescent thermophoresis signal were plotted against the concentration of the serially diluted MHV spike protein, and Kd values were determined using the NanoTemper analysis software.
4
Quantifying Spx-YjbH Binding via MST
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Binding between Spx and YjbH was measured by MST. Spx was labelled with the Monolith Protein Labeling Kit (NanoTemper Technologies, Munich, Germany) using red fluorescent dye NT-647 NHS (amine-reactive) according to the manufacturer's instructions. Labelling reagents were removed by buffer-exchange column chromatography, and labelled Spx was eluted in PBS with 0.05% Tween-20. Binding assays were performed with a Monolith NT.115 device (Nanotemper Technologies, Munich, Germany). Fluorescently labelled Spx (4.3 nM) was incubated for 10 min at RT with 2-fold serial dilutions of YjbH (14 concentrations from 0.1 mM to 2 nM) in 50 mM potassium phosphate buffer (pH 7.5), 150 mM NaCl. The samples were loaded into premium coated capillaries (MonolithÔ NT.115 MST Premium Coated Capillaries). MST traces were recorded at 20 C in a Monolith NT.115 (NanoTemper Technologies, Munich, Germany) using the MO.Control software. After recording MST-traces, the normalized fluorescence, F norm , defined as the ratio between the fluorescence after and before heating with an infrared laser, was plotted against the concentration of YjbH.
5
VP1 Binding Affinity Determination
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To investigate whether PTC-209HBr binds to VP1, an MST assay was carried out to determine the binding affinities between VP1 and the compounds. Compounds were diluted to the indicated concentrations and mixed with VP1-EGFP in assay MST buffer containing 0.05% Tween 20. Then, the mixed samples were loaded into Monolith NT.115 capillaries, and thermophoresis was analyzed by a Monolith NT.115 instrument (NanoTemper Technologies). The thermophoresis parameters were set as blue excitation with a power at 20% in LED, “medium” in MST power and an optimized time setting (2.5 s, MST on) as reported (Entzian and Schubert, 2016 (link)). GraphPad Prism 8 software was used out to calculating Kd value by fitting the MST data.
6
Kelch Domain Fluorescence Measurement
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For MST measurements, the Kelch domain was fluorescently labeled with RED-MALEIMIDE (Cysteine reactive) fluorescent dye (NanoTemper, München, Germany), and Nano-RED excitation (20%) was used. Measurements were carried out on Monolith NT.115 (NanoTemper, München, Germany) at 25 °C in 50 mM Tris, 100 mM NaCl, pH 8.2, 0.05% Tween buffer, in Monolith NT.115 capillaries. The concentration of the labeled Kelch protein was 50 nM.
7
Fluorescent Labeling of Transgelin Protein
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The fluorescence labeling of transgelin was performed following the protocol for the N-hydroxysuccinimide (NHS) coupling of dye NT647 (NanoTemper Technologies, Munich, Germany) to lysine residues. In brief, 100 μL of a 20 μM solution of transgelin protein in labeling buffer was mixed with 100 μL of 60 μM NT647-NHS fluorophore (NanoTemper Technologies) in labeling buffer and was incubated for 30 min at room temperature (RT). Pretests using premium-coated and standard-treated MST capillaries (NanoTemper Technology) were performed to test for the adsorption of NT647 MEK1 to the capillary walls by analyzing capillary scans recorded by Monolith NT.115 prior to the MST experiments. Interactions observed between salvianolic acid A and NT647-transgelin were established on a Monolith NT.115 instrument (NanoTemper Technologies, Germany) and were used as a positive control during screening. For this purpose, serial dilutions of salvianolic acid A in assay buffer were prepared and mixed 1:1 with a solution of 30–50 nM NT647-transgelin to yield a final volume of 20 μL per dilution. The reaction mixtures were loaded into standard-treated capillaries and then analyzed by MST at 20% and 80% MST power and at a light-emitting diode (LED) intensity of 30%.
8
Protein-Ligand Binding Affinity by MST
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Purified His-Cry5B(27–698) and His-Cry5B(112–698) as protein targets were labelled using Alexa Fluor 647 NHS ester dye according to the instruction. Unreacted dye was removed with a desalting column (30K MWCO, Bio-Rad). The labelled targets were adjusted to appropriate concentrations for detection, and the galactose as ligand was freshly solubilized in the same buffer—aforementioned buffer B (20 mM HEPES pH 8.0, 50 mM NaCl).
For each assay, 16 different serially-diluted concentrations of ligand were firstly prepared, and then mixed with equal volume of labelled protein target at room temperature. The reaction mixtures were loaded into standard Monolith NT.115 capillaries and measured using a Monolith NT.115 instrument (NanoTemper Technologies). Instrument parameters were adjusted to 40% MST power and 2% or 6% excitation power (2% for Cry5B(27–698) and 6% for Cry5B(112–698). The Kd values were calculated using MO.Affinity Analysis v.2.2.4 software (NanoTemper Technologies) as mean ± SEM from at least three independent experiments with a single site-specific binding model.
For each assay, 16 different serially-diluted concentrations of ligand were firstly prepared, and then mixed with equal volume of labelled protein target at room temperature. The reaction mixtures were loaded into standard Monolith NT.115 capillaries and measured using a Monolith NT.115 instrument (NanoTemper Technologies). Instrument parameters were adjusted to 40% MST power and 2% or 6% excitation power (2% for Cry5B(27–698) and 6% for Cry5B(112–698). The Kd values were calculated using MO.Affinity Analysis v.2.2.4 software (NanoTemper Technologies) as mean ± SEM from at least three independent experiments with a single site-specific binding model.
9
Binding Affinity of BSpep to Cy5-α-BgTx
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Binding of BSpep to Cy5-α-BgTx was investigated by microscale thermophoresis (MST) using the Monolith NT 115 (NanoTemper Technologies GmbH, Germany). All dilutions were performed in proprietary MST NT.115 buffer, supplemented with 0.05% Tween 20. Briefly, 5 nM of Cy5-α-bungarotoxin was incubated with a range of BSpep concentrations for 15 min at room temperature, before being loaded in Monolith Premium capillaries (NanoTemper Technologies GmbH, Germany) and mounted in the Monolith NT 115 thermophoresis chamber. MST was monitored using the dedicated MO Control software. Readings were performed using the built-in picoRED illumination source (600 to 650 nm) at 20% illumination power, and MST power was set as medium. MST fluorescence (F1) was averaged from 1.5 to 2.5 min through MST and normalised against initial fluorescence (F0), which was recorded 1 min before the start of MST. The resulting normalised fluorescence values were expressed as the difference from initial fluorescence (ΔFnorm), which was plotted against BSpep concentration. Dose-ΔFnorm relationships were fit to a sigmoidal equation: Y = Bottom + (Top − Bottom)/(1 + 10^((LogIC50 − X) × HillSlope)).
10
Quantifying Env-Antibody Binding Kinetics
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Ligand binding to purified Env-NDs was analyzed by microscale thermophoresis [84 (link)] on a Monolith NT.115 (Nanotemper Technologies). The glutaraldehyde-crosslinked HIV-1JR-FL Env (-)Δ808-ND sample was fluorescent, precluding the need for labeling with an additional fluorophore. The binding of the 2G12 antibody to the Env(-)Δ808-NDs was tested using antibody concentrations ranging from 0.061 nM to 2 μM, in 20 mM Tris-HCl pH 7.5, 300 mM NaCl. The glutaraldehyde-crosslinked Env(-)Δ808-ND sample was added to a final constant concentration of 20 nM, followed by a 1-hr incubation at room temperature. The Env(-)Δ808-ND concentration was chosen such that the observed fluorescence was approximately 1,000 U at 50% LED power. The samples were loaded into Monolith capillaries and were measured by standard protocols of temperature shifts using a Monolith NT.115 (NanoTemper) as described by the manufacturer. The changes in the fluorescent thermophoresis signal were plotted against the concentration of the serially diluted antibody, and Kd values were determined using the NanoTemper analysis software.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!