Monolith nt 115 capillaries

Manufactured by NanoTemper

Sourced in Germany

The Monolith NT.115 Capillaries are a core piece of lab equipment designed for biophysical analysis. They provide a controlled environment for sample measurement, enabling high-precision data collection. The capillaries are compatible with the NanoTemper Monolith instrument and facilitate the analysis of biomolecular interactions and conformational changes.

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29 protocols using monolith nt 115 capillaries

Mupirocin Binding Kinetics to IleRS

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MST assays were performed with a Monolith NT.115 instrument (NanoTemper
Technologies, Germany) (Duhr and Braun,
2006; Seidel et al., 2013 (link)).
Each titration curve con- tained 16 points prepared by serial dilutions of
analytes and a constant concentration of the fluorescein-labeled ligand. To
measure the binding affinity between mupirocin and purified human IleRS and
Ca IleRSs (wild-type and mutants), proteins were
fluorescently labeled using a Monolith His-Tag Labeling Kit RED-TRIS-NTA 2nd
Generation (NanoTemper Technolo- gies). A 2 mM sample of mupirocin was serially
diluted 2-fold by 15 times and then the 16 different concentrations of mupirocin
were incubated with 400 nM of labeled human IleRS or Ca IleRS
(wild-type or mutants). Experiments were performed in HEPES buffer supplemented
with 0.05% (w/v) Tween-20 and 0.05% bovine serum albumin (BSA). Samples were
loaded into high-precision Monolith NT.115 Capillaries (NanoTemper
Technologies). MST assays were performed with 60% light-emitting diode (LED)
power using a green filter and 60% MST power. Normalized fluorescence read- ings
(thermophoresis plus T-jump) were plotted as a function of analyte
concentration, and curve fitting and dissociation constant (K_d)
calculation were performed with MO.Affinity Analysis (ver. 2.3; NanoTemper
Technologies). For each data set, three independent MST measurements were
carried out.

Chung S., Kim S., Ryu S.H., Hwang K.Y, & Cho Y. (2020). Structural Basis for the Antibiotic Resistance of Eukaryotic Isoleucyl-tRNA Synthetase. Molecules and Cells, 43(4), 350-359.

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Fluorescent Labeling and Binding Assay

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The Monolith NT™ Protein Labelling Kit Red-NHS containing the red fluorescent dye NT-647 was used to label recombinant EPAC1-CNBD protein according to the manufacturer’s protocol. Equal volumes of labelled protein were then added to Monolith NT.115 capillaries (Nanotemper technologies) and then measured at LED/excitation set at 80–100% and MST power of 60% (high setting) on the MST instrument. The normalized fluorescence (Fnorm (%)) from MO.Affinity Analysis software version 2.2.4 (Nanotemper technologies) was normalized as a fold change with the relative fluorescence associated with the lowest concentration of antibody set to 1 (Arbitrary Units: AU). The pEC50 was determined by nonlinear curve fitting analysis (Log (agonist) vs. response (three parameters)) GraphPad Prism.

Buist H.K., Luchowska-Stańska U., van Basten B., Valli J., Smith B.O., Baillie G.S., Rickman C., Ricketts B., Davidson A., Hannam R., Sunderland J, & Yarwood S.J. (2021). Identification and Characterization of an Affimer Affinity Reagent for the Detection of the cAMP Sensor, EPAC1. Cells, 10(9), 2307.

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Microscale Thermophoresis of GlyR

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Microscale thermophoresis (MST) analysis was performed using a NanoTemper Monolith NT.115 instrument (NanoTemper Technologies, Munich, Germany). Therefore, purified α1-His GlyR nanodiscs or total solubilized cell membranes with α1-GFP GlyR were diluted to a concentration of 400 nM in PBS. α1-His GlyR was fluorescence-labeled using the Monolith NT His-Tag Labeling Kit RED-tris-NTA (NanoTemper Technologies). Labeled GlyR α1 SMALPs were added in a 1:1 ratio to a 1:2 dilution series with a final concentration of 3 mM down to 0.73 µM for glycine or 12.5 mM down to 6 µM for taurine, as well as 0 µM for each ligand as an internal control and loaded into standard capillaries (Monolith NT.115 Capillaries, NanoTemper Technologies). Thermophoresis was measured at 21 °C for 15 or 20 s with 40% LED power and 60% infrared laser power. For MST experiments n = 3–4 independent technical measurements were collected from N = 2–3 independent oocyte or HEK293 cell batches.

Bernhard M, & Laube B. (2020). Thermophoretic analysis of ligand-specific conformational states of the inhibitory glycine receptor embedded in copolymer nanodiscs. Scientific Reports, 10, 16569.

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Protein Interaction Analysis by Pull-down and MST

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The pull-down assay was performed following the method of our previous study [99 (link)], briefly, the indicated proteins were fused with 6×His or GST tags and expressed in Escherichia coli strain BL21. The supernatant containing 6×His- and GST-tagged soluble proteins was mixed and incubated with BeaverBeads IDA-Nickel (Beaver Biosciences, China) at 4°C for 3 h. The beads were collected and washed four times with Tris buffer. The eluted proteins were separated by SDS-PAGE and detected using anti-His and anti-GST antibodies.
MST was performed as previously described [99 (link), 100 ]. CaWRKY40-GFP, CabZIP63-GFP, CaTAF14b-GFP, CaRUVBL2-GFP fusion protein, or GFP (as a control) was used for the fluorescent label and CaSWC4-6×His fusion protein was used for the non-fluorescent label as described by Huang et al. [98 (link)]. CaSWC4-6×His fusion protein was diluted to a range of concentrations from 1.0E^–10 mM to 1.0E^–3 mM, then incubated with 20 mM of the labeled protein for 10 min in interaction buffer. The samples were then loaded into Monolith NT.115 Capillaries (Cat#MO-K002, NanoTemper Technologies, Germany) using 50% IR laser power and an LED excitation source, where λ = 470 nm at room temperature. We used the Nano Temper Analysis 1.2.20 software to fit the data and determine apparent K_d values [101 (link), 102 (link)].

Cai W., Yang S., Wu R., Zheng Y., He S., Shen L., Guan D, & He S. (2022). CaSWC4 regulates the immunity-thermotolerance tradeoff by recruiting CabZIP63/CaWRKY40 to target genes and activating chromatin in pepper. PLoS Genetics, 18(2), e1010023.

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Microscale Thermophoresis Binding Assay

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Recombinantly purified protein constructs were labelled with RED-tris NTA dye (NanoTemper Technologies GmbH) in PBS to a concentration of 100 nM. A 20 µM stock of non-labelled protein was also prepared. This stock was used in a 16-step serial dilution in PBS buffer. For oligomerisation studies,10 µM of protein was used as the highest ligand concentration, with Red-tris-NTA labelled protein kept at a final concentration of 50 nM for all reactions.
Reactions were incubated for 15 min at room temperature in the dark and loaded into Monolith NT.115 Capillaries (NanoTemper Technologies GmbH). Microscale thermophoresis measurements were performed using a Monolith NT.115 (NanoTemper Technologies GmbH) with 20%(RED) LED and high MST power. Binding assays were run as three independent experiments and the data were fitted using a K_D model with ligand-induced initial fluorescence change, as described by Jerabek-Willemsen et al.^{49 (link)}.

dos Santos Á., Rollins D.E., Hari-Gupta Y., McArthur H., Du M., Ru S.Y., Pidlisna K., Stranger A., Lorgat F., Lambert D., Brown I., Howland K., Aaron J., Wang L., Ellis P.J., Chew T.L., Martin-Fernandez M., Pyne A.L, & Toseland C.P. (2023). Autophagy receptor NDP52 alters DNA conformation to modulate RNA polymerase II transcription. Nature Communications, 14, 2855.

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Quantitative Spike-ACE2 Binding Assay

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Spike RBD proteins were labeled using Monolith His-Tag Labeling Kit RED-tris-NTA (NanoTemper Technologies) following manufacturer protocol at 2:1 protein to dye ratio. Briefly, 100 uL of 200nM Spike RBD (Hu-1 or B.1.1.7 or B.1.351 RBD) was mixed with 100 uL of 100nM dye for 30 minutes at room temperature, followed by centrifugation for 10 minutes at 15000g at 4C. A series of sixteen 1:1 dilutions of hACE2 were prepared, with 2.27uM the highest concentration. Each dilution of hACE2 was mixed with one volume of Spike labeling reaction mix prior to incubation for 5 minutes at room temperature. Mixed samples were loaded into Monolith NT.115 Capillaries (NanoTemper Technologies). As a specificity control, hnRNPC was utilized instead of hACE2; MST showed no binding (Figure S6). MST was performed using a Monolith NT.115 instrument (NanoTemper Technologies) at room temperature, with 60–80% excitation power and Medium MST power.

Ramanathan M., Ferguson I.D., Miao W, & Khavari P.A. (2021). SARS-CoV-2 B.1.1.7 and B.1.351 Spike variants bind human ACE2 with increased affinity. bioRxiv.

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Thermophoretic Binding Assay of AMP-PNP to Hsp90

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HSP90-mNeonGreen (C. albicans) or mNeonGreen serving as the negative control was adjusted to 100 nM with Sba1 buffer (40 mM HEPES/KOH pH 7.5, 20 mM KCl). The ligand AMP-PNP was dissolved in 2x reaction buffer (40 mM HEPES/KOH pH 7.5, 20 mM KCl, 10 mM MgCl₂, 40 mM Na₂MoO₄, 0.2% Triton-X 100), and a series of 16 1:1 dilutions was prepared using the same buffer, producing ligand concentrations ranging from 305 to 10 000 nM. For the measurement, each ligand dilution was mixed with one volume of HSP90-mNeonGreen or mNeonGreen, which led to a final concentration of 50 nM and final ligand concentrations ranging from 153 nM to 5 mM. After 5 min incubation at room temperature, the samples were loaded into Monolith NT.115 Capillaries (NanoTemper Technologies, Munich, Germany). MST was measured using a Monolith NT.115 at an ambient temperature of 25 °C. Instrument parameters were adjusted to 35% LED power and a high MST power of 80%. Data of three independently pipetted measurements were analyzed (MO.Affinity Analysis software version 2.1.3, NanoTemper Technologies, Munich, Germany) using the signal from an MST-on time of 30 s.

Kohlmann P., Krylov S.N., Marchand P, & Jose J. (2024). FRET Assays for the Identification of C. albicans HSP90-Sba1 and Human HSP90α-p23 Binding Inhibitors. Pharmaceuticals, 17(4), 516.

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EGFR Peptide Binding Affinity with Kindlin-1

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Recombinantly expressed kindlin-1 constructs were coupled to an equimolar amount of RED-tris-NTA NT-647 dye (NanoTemper Technologies, München, Germany) via its N-terminal 6×His-Tag in a one-step coupling reaction (Bartoschik et al., 2018 (link)). Titrations were performed in PBS (137 mmol/L NaCl, 27 mmol/L KCl, 100 mmol/L Na₂HPO₄, 18 mmol/L KH₂PO₄) using a constant 50-nmol/L concentration of RED-tris-NTA–coupled kindlin, with increasing concentration of synthetic EGFR peptide (residues 668-711: CMRRRHIVRKRTLRRLLQERELVEPLTPSGEAPNQALLRILKETE) and final volume of 20 μl. Prepared samples were filled into Monolith NT.115 Capillaries (NanoTemper). Measurements were recorded on a Monolith NT.115 at 25°C, excited under red light, medium MST power, and 40% excitation power. The data were analyzed using MO Affinity Analysis software (NanoTemper) and fitted using the K_d fit model.

Michael M., Begum R., Chan G.K., Whitewood A.J., Matthews D.R., Goult B.T., McGrath J.A, & Parsons M. (2019). Kindlin-1 Regulates Epidermal Growth Factor Receptor Signaling. The Journal of Investigative Dermatology, 139(2), 369-379.

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Determining eIF4E-capped RNA Binding Kinetics

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To determine the dissociation constant for eIF4E protein and capped short RNA complexes, an MST-based method was applied, previously described in (28 (link)), with modification of the utilized ligand form; here, capped short RNAs were analysed. Binding assay mixtures were prepared in 20 μl as follows: 10 nM fluorescent probe m⁷Gp₅OC₃(5)FAM, 50 nM murine eIF4E and ligand (differently capped short RNAs ranging from 1.75 μM to 0.05 nM) in an MST buffer (50 mM HEPES–KOH pH 7.2, 100 mM KCl, 0.5 mM EDTA, 0.05% Tween-20). Samples after preparation, without any additional incubation, were loaded into Monolith NT.115 Capillaries (MO-KO22; NanoTemper Technologies). MST was performed using a Monolith NT.115 instrument (NanoTemper Technologies) at 25°C. Instrument parameters were adjusted to 100% LED Blue power and Medium MST power. To determine the K_D_,app values, a standard 1:1 binding model was fitted to the data using PALMIST software (version 1.4.4). Confidence intervals were determined using error-surface projection (ESP) (29 (link)).

Drazkowska K., Tomecki R., Warminski M., Baran N., Cysewski D., Depaix A., Kasprzyk R., Kowalska J., Jemielity J, & Sikorski P.J. (2022). 2′-O-Methylation of the second transcribed nucleotide within the mRNA 5′ cap impacts the protein production level in a cell-specific manner and contributes to RNA immune evasion. Nucleic Acids Research, 50(16), 9051-9071.

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Measuring CaHSF8-CaKAN3 Protein Interaction

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The MST assay was used to confirm the protein‒protein interaction as described in previous studies^{107 (link)}. To confirm the protein interaction between CaHSF8-6×his and CaKAN3-GST, the two fused proteins were prokaryotically expressed, and CaKAN3-GST was marked by fluorescence (Mo-L011, NanoTemper Technologies, Germany). CaHSF8-6×his was diluted to concentrations ranging from 1.0E-10 mM to 1.0E-3 mM and mixed with 20 mM GST or CaKAN3-GST protein solution. The mixtures were incubated with an interaction buffer (100 mM NaCl, 1 mM EDTA, 20 mM sodium phosphate, pH 8.0). The samples were then loaded into Monolith NT.115 Capillaries (MO-K002, NanoTemper Technologies, Germany) using 50% IR laser power and an LED excitation source, where λ = 470 nm at ambient temperature. The Kd values for the protein interactions were calculated using NanoTemper Analysis 1.2.20 software^{108 (link)}.

Yang S., Cai W., Wu R., Huang Y., Lu Q., Hui W.a.n.g., Huang X., Zhang Y., Wu Q., Cheng X., Wan M., Lv J., Liu Q., Zheng X., Mou S., Guan D, & He S. (2023). Differential CaKAN3-CaHSF8 associations underlie distinct immune and heat responses under high temperature and high humidity conditions. Nature Communications, 14, 4477.

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