Protein labeling kit red nhs 2nd generation

Manufactured by NanoTemper

Sourced in Germany

The Protein Labeling Kit RED-NHS 2nd Generation is a lab equipment product designed for labeling proteins. It provides a toolkit for covalently attaching a red fluorescent dye to proteins, enabling visualization and detection.

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Lab products found in correlation

Standard capillaries, by NanoTemper (2 mentions) Monolith mst device, by NanoTemper (2 mentions) Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions) Monolith nt 115, by NanoTemper (2 mentions) Eclipse ti2 microscope, by Nikon (1 mentions) Monolith nt 115 blue red, by NanoTemper (1 mentions) Monolith nt 115 instrument, by NanoTemper (1 mentions) Bl21 codonplus de3 ril competent cells, by Agilent Technologies (1 mentions) Precision xs microplate sample processor, by Agilent Technologies (1 mentions) Monolith nt automated capillary chips, by NanoTemper (1 mentions) Mo affinity analysis software, by NanoTemper (1 mentions) Nt 115, by NanoTemper (1 mentions) Nt 115 instrument, by NanoTemper (1 mentions)

Spelling variants of this product

RED-NHS protein labeling kit (19 citations) RED-NHS (13 citations) Monolith Protein Labeling Kit RED-NHS 2nd Generation (11 citations) RED-NHS labeling kit (7 citations) RED-NHS 2nd Generation (6 citations) RED-NHS 2nd Generation kit (3 citations) Protein Labeling Kit (2 citations) Monolith NT™ Protein Labeling Kit RED-NHS 2nd Generation (2 citations) Monolith Protein Labeling Kit RED-NHS 2nd Generation (Amine Reactive) (2 citations)

13 protocols using protein labeling kit red nhs 2nd generation

Probing SARS-CoV-2 N-Protein-RNA Interactions

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Recombinant SARS-CoV-2 N-protein with His-tag, obtained as described previously [27 (link)] (purity: ≥90%), was a kind gift of the group of Prof. V. N. Lazarev. The protein was labeled with a far-red-emitting RED dye using the RED-NHS 2nd Generation Protein Labeling Kit (Nanotemper, Munich, Germany). The stem-loop fragment of SARS-CoV-2 gRNA (SL5 in [43 (link)]) was obtained by in vitro transcription from the T7-promoter-containing dsDNA template according to the published procedure [27 (link)].
The labeled N-protein (3 µM final concentration) was mixed with RNA (6 µM final concentration) in the RNAse-free 20 mM sodium phosphate buffer (pH 6) supplemented with 150 mM NaCl. Compound 3a/3b/7a/9a was added to a final concentration of 20 µM. The mixture was incubated at 37 °C for 2 h. Then, 3 μL of the mixture was placed between glass slides and analyzed using a Nikon Eclipse Ti2 microscope (Nikon, Tokyo, Japan) in the red channel. All experiments were performed in 6 repeats. Droplets were counted in ImageJ software, version 1.53a, and the partitioning coefficient (PC = [N-protein]_droplet/[N-protein]_solution) was calculated based on the average fluorescence intensity values in droplets and bulk solution.

Kamzeeva P., Petushkov I., Knizhnik E., Snoeck R., Khodarovich Y., Ryabukhina E., Alferova V., Eshtukov-Shcheglov A., Belyaev E., Svetlova J., Vedekhina T., Kulbachinskiy A., Varizhuk A., Andrei G, & Aralov A. (2023). Phenotypic Test of Benzo[4,5]imidazo[1,2-c]pyrimidinone-Based Nucleoside and Non-Nucleoside Derivatives against DNA and RNA Viruses, Including Coronaviruses. International Journal of Molecular Sciences, 24(19), 14540.

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Protein-Ligand Binding Affinity Assay

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Purified SR-B1 or CD36 protein was labeled using the RED-NHS 2nd Generation Protein Labeling Kit (NanoTemper Technologies) according to manufacturer’s instructions. Samples were prepared in exchange buffer and loaded into standard capillaries. Measurements were performed on the Monolith NT.115 BLUE/RED (NanoTemper Technologies) at 25 °C using 40% MST power and laser on/off times of 0 s and 21 s, respectively. Labeled receptor (SR-B1 or CD36) was diluted in exchange buffer to a concentration of 20 nM. For each ligand, 10 μl of starting concentration ligand was diluted 1:1 in 10 μl of exchange buffer to make a 16-sample dilution series. Ten microliters of receptor at constant concentration (20 nM) was incubated with 10 μl of ligand at each dilution in the series, briefly centrifuged, and loaded into Monolith NT.115 capillaries. Each sample was checked for sample aggregation and capillary adsorption and points with either were removed. All measurements were performed with protein from two separate protein purifications. The MST technique is described in further detail (27 ). Apparent K_d values were calculated by nonlinear regression analysis, assuming one-site specific binding, of the normalized thermophoresis (∂Fnorm), using GraphPad Prism (https://www.graphpad.com/).

Powers H.R., Jenjak S.E., Volkman B.F, & Sahoo D. (2023). Development and validation of a purification system for functional full-length human SR-B1 and CD36. The Journal of Biological Chemistry, 299(10), 105187.

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Labeling and Quantification of AAG Protein

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Native and desialylated AAG were labeled using the RED-NHS 2nd Generation protein labeling kit (NanoTemper Technologies GmbH, Munich, Germany). The labeling reaction was performed according to the manufacturer’s instructions in the supplied labeling buffer (130 mM NaHCO₃, 50 mM NaCl, pH 8.2–8.3 at room temperature) by applying 90 µL of 10 µM AAG (molar dye:AAG = 3:1) at room temperature for 30 min. The labeled protein was purified using the B-column provided by the labeling kit. The AAG concentration c_AAG and the degree of labeling (DOL) were calculated using Equations (3) and (4):

c_{A A G} (M) = \frac{A_{280} - (A_{650} \times 0.04)}{ε_{A G P 280} \times d}

D O L = \frac{A_{650}}{195 \times 10^{3} M^{- 1} {c m}^{- 1} \times c_{A A G} (M)}

where

ε_{A A G 280}

is the AAG molar absorption coefficient at 280 nm, d is path length of a spectrophotometer in cm, and 0.04 is a correction factor at 280 nm. Aliquoted and flash-frozen labeled protein was stored in liquid nitrogen.

Šeba T., Kerep R., Weitner T., Šoić D., Keser T., Lauc G, & Gabričević M. (2024). Influence of Desialylation on the Drug Binding Affinity of Human Alpha-1-Acid Glycoprotein Assessed by Microscale Thermophoresis. Pharmaceutics, 16(2), 230.

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Microscale Thermophoresis of NSP10

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For microscale thermophoresis (MST) assays, NSP10 protein was fluorescently labeled using N-Hydroxysuccinimide (NHS)-ester fluorophores according to the manufacturer’s instructions (Protein Labeling Kit RED-NHS 2nd Generation, Nanotemper). Labeling was performed in the NSP16 protein storage buffer (20 mM HEPES pH 7.5 and 0.3 M NaCl). Prior to use in MST experiments, the labeled protein solution (2 µM) was centrifuged at 15.000 × g for 10 min at 4 °C to remove potential aggregated protein species. Compound/NSP10 experiments were performed in 20 mM HEPES, 0.3 M NaCl with 0.05% Tween. For the binding studies, 100 or 200 nM NSP10-RED (depending on labeling efficiency) were incubated with increasing amounts of compound for 15 min at RT. Measurements were performed in standard capillaries (Nanotemper) using a Monolith MST device (Nanotemper) with 80% LED and medium infrared (IR) laser power. Binding was analyzed from MST signals after 5 or 20 s compared to relative fluorescence before IR laser pulse.

Trepte P., Secker C., Olivet J., Blavier J., Kostova S., Maseko S.B., Minia I., Silva Ramos E., Cassonnet P., Golusik S., Zenkner M., Beetz S., Liebich M.J., Scharek N., Schütz A., Sperling M., Lisurek M., Wang Y., Spirohn K., Hao T., Calderwood M.A., Hill D.E., Landthaler M., Choi S.G., Twizere J.C., Vidal M, & Wanker E.E. (2024). AI-guided pipeline for protein–protein interaction drug discovery identifies a SARS-CoV-2 inhibitor. Molecular Systems Biology, 20(4), 428-457.

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Binding Affinity of CALR del52 to TpoR

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Recombinant human CALR del52 was labeled with NHS- chemistry according to the manufacture’s instruction (Protein Labeling Kit RED-NHS 2^nd Generation, NanoTemper Technology), referred as the “target protein”. Briefly, the target protein (at 10 μM) was incubated with the dye solution in the labeling buffer (130 mM NaHCO₃, 50 mM NaCl, pH 8.2–8.3) for 1 h on ice. The dye carries a reactive NHS-ester group that reacts with primary amines (lysine residues) to form covalent bonds. The surplus of the dye not bound to the target protein was removed through passage on a resin column prior to elution of the target in the equilibration buffer (Tris-HCL 1 M, pH 7.6). For MST measurement, the CALR del52-NHS was used at 20 nM final concentration in MST buffer (Tris based supplemented with 0.01% of tween 20).
TpoR D1–D4 (the “ligand”) remained label free. Serial dilutions (0.15 nM to 5 μM) of the ligand (TpoR D1-D4) were performed to titer the target protein (CALR del52). The measurements were performed on a NanoTemper monolith NT.115 instrument (NanoTemper technologies, Germany) at 40% LED and medium MST-power with a standard 5 s. before, MST-on for 30 s. and 5 s. after MST-off.

Papadopoulos N., Nédélec A., Derenne A., Şulea T.A., Pecquet C., Chachoua I., Vertenoeil G., Tilmant T., Petrescu A.J., Mazzucchelli G., Iorga B.I., Vertommen D, & Constantinescu S.N. (2023). Oncogenic CALR mutant C-terminus mediates dual binding to the thrombopoietin receptor triggering complex dimerization and activation. Nature Communications, 14, 1881.

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SARS-CoV-2 Mpro Protein Labeling Protocol

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The concentration of the purified SARS-CoV-2 Mpro protein was measured using the NanoDrop Spectrophotometer and BCA assay (Pierce™ BCA Protein Assay Kit, Thermo Fisher Scientific, Rockford, IL, USA) based on the beforehand prepared calibration curve on the bovine serum albumin. In the next step, a part of the Mpro protein was labeled using the Protein Labeling Kit RED-NHS 2nd Generation (NanoTemper Technologies, Munich, Germany) according to the manufacturer’s instructions. The concentration of the protein in the labeling mixture was adjusted to 10 μM and the molar dye:protein ratio was 3:1. The labeling reaction was performed in the labeling buffer NHS (130 mM NaHCO₃, 50 mM NaCl, pH 8.2–8.3) at room temperature for 30 min in the dark. The unbound dye was removed using a dye removal column equilibrated with HEPES buffer. The degree of labeling (DOL parameter) was determined using UV/VIS spectrophotometry at 650 and 205 nm. The achieved DOL value was around 1.0. After the labeling, the labeled protein solution was supplemented with Pluronic F-127 with the final concentration of 0.01% (w/v).

Papaj K., Spychalska P., Hopko K., Kapica P., Fisher A., Lill M.A., Bagrowska W., Nowak J., Szleper K., Smieško M., Kasprzycka A, & Góra A. (2021). Investigation of Thiocarbamates as Potential Inhibitors of the SARS-CoV-2 Mpro. Pharmaceuticals, 14(11), 1153.

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PD-L1 Binding Affinity Determination

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PD-L1 was expressed
as for NMR experiment. It was diluted to 10 μM and labelled
with Protein Labeling Kit RED-NHS 2nd Generation from Nanotemper according
to the manufacturer’s guidelines. The degree of labelling for
PD-L1 was 0.74, which is with agreement with the protocol. Labelled
PD-L1 at 100 nM concentration was then mixed with independent series
of 4a and 9a compound dilution series (n = 4) and incubated for 30 min at RT. Next, samples were
transferred to dedicated 384-well plate and measured on Diantus equipped
with pico detector using auto-excitation and 5 s pulse (Nanotemper)
for the affinity measurements. The results were exported, averaged,
and fitted in Mathematica 12 using following equations: where: FB—fraction bound, [A]—concentration
of unlabeled titrated partner, [B]—concentration of fluorescent
labeled partner that is fixed, [AB]—concentration of bound
complex of A and B, K_d—equilibrium
dissociation constant.

Ważyńska M.A., Butera R., Requesens M., Plat A., Zarganes-Tzitzikas T., Neochoritis C.G., Plewka J., Skalniak L., Kocik-Krol J., Musielak B., Magiera-Mularz K., Rodriguez I., Blok S.N., de Bruyn M., Nijman H.W., Elsinga P.H., Holak T.A, & Dömling A. (2023). Design, Synthesis, and Biological Evaluation of 2-Hydroxy-4-phenylthiophene-3-carbonitrile as PD-L1 Antagonist and Its Comparison to Available Small Molecular PD-L1 Inhibitors. Journal of Medicinal Chemistry, 66(14), 9577-9591.

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Microscale Thermophoresis Assay for Protein-RNA Interactions

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All microscale thermophoresis measurements (MST) were performed on a NanoTemper Monolith NT.115 instrument (NanoTemper Technologies, Munich, Germany). using the Standard Treated Capillaries K002 of the supplier. Each of the 16 solutions of one titration series was filled into a capillary, which were measured successively to create the respective data points in the experiment. General settings were applied for all MST experiments as follows: manual temperature control: 22°C, LED laser: RED. MST power is set at medium and excitation power is set at 20%. For MST assay, all the proteins were exchanged into the MST buffer (20 mM HEPES pH7.5, 500 mM NaCl, 5% glycerol). The TPR-CHAT and its mutants were fluorescence-labeled using the Protein Labeling Kit RED-NHS 2nd Generation (NanoTemper Technologies) at 10 μM. 50 nM fluorescence-labeled TPR-CHAT and its mutants were added in a 1:1 ratio to a 1:2 dilution series with a final concentration of 2 μM down to 0.122 nM for gRAMP-crRNA complex. Each experiment was conducted at least 3 times and the similar result was obtained each time. Each protein K D value was obtained with a signal-to-noise ratio higher than 10. Datasets were processed with the MO (Monolith). Affinity Analysis v2.3 software. The analysis of dose-response curves was carried out with OriginPro 2018.

Liu X., Zhang L., Wang H., Xiu Y., Huang L., Gao Z., Li N., Li F., Xiong W., Gao T., Zhang Y., Yang M, & Feng Y. (2022). Target RNA activates the protease activity of Craspase to confer antiviral defense. Molecular cell, 82(23).

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Thermophoretic Characterization of RORγt Binding

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For MST screening, we used Monolith NT.115 instrument from Nanotemper to assess the compounds/RORγt interaction. We procured recombinant human RORγt from WuXi and labeled it with Protein Labeling Kit RED-NHS 2nd Generation from Nanotemper (Cat #MO-L011). RORγt was produced by recombinant microbial expression from E. coli cells (BL21-CodonPlus (DE3)-RIL Competent Cells, Cat #230245 from AGILENT). We dissolved RORγt in PBS buffer (pH 7.4) with 0.1% bovine serum albumin (BSA) and 0.05% Tween 20. We kept the concentration of the fluorescently labeled RORγt constant at 25 nM. We added a volume of 5 μL of the corresponding samples in MST capillaries with a final DMSO concentration of 2%. Subsequently, we incubated the samples within the capillaries for 20 min at room temperature prior to the measurements. We detected changes in thermophoretic properties as a change in fluorescence intensity upon incubation of various concentrations of the tested compounds with fluorescently labeled RORγt. We plotted the thermophoresis signal against the compound concentration to obtain a dose–response curves, from which K_D values can be deduced. MST data are represented as normalized change in fluorescence (F_norm) upon ligand binding. F_norm is the ratio of the fluorescence measured before and during thermophoresis.

Abdel-Rahman S.A., Brogi S, & Gabr M.T. (2024). Lithocholic acid derivatives as potent modulators of the nuclear receptor RORγt. RSC Advances, 14(5), 2918-2928.

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Microscale Thermophoresis of NSP10

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For microscale thermophoresis (MST) assays, NSP10 protein was fluorescently labeled using N-Hydroxysuccinimide (NHS)-ester fluorophores according to the manufacturer’s instructions (Protein Labeling Kit RED-NHS 2nd Generation, Nanotemper). Labeling was performed in the NSP16 protein storage buffer (20 mM HEPES pH 7.5 and 0.3 M NaCl). Prior to use in MST experiments, the labeled protein solution (2 μM) was centrifuged at 15.000xg for 10 min at 4°C to remove potential aggregated protein species. NSP16/NSP10 MST experiments were performed in 20 mM HEPES, 0.3 M NaCl with 0.5% Tween, compound/NSP10 experiments in 20 mM HEPES, 0.3 M NaCl with 0.05% Tween. For the binding studies, 100 or 200 nM NSP10-RED (depending on labeling efficiency) were incubated with increasing amounts of NSP16 or compound for 15 min at RT. Measurements were performed in standard capillaries (Nanotemper) using a Monolith MST device (Nanotemper) with 80% LED and medium infrared (IR) laser power. Binding was analyzed from MST signals after 5 or 20 s compared to relative fluorescence before IR laser pulse.

Trepte P., Secker C., Kostova S., Maseko S.B., Choi S.G., Blavier J., Minia I., Ramos E.S., Cassonnet P., Golusik S., Zenkner M., Beetz S., Liebich M.J., Scharek N., Schütz A., Sperling M., Lisurek M., Wang Y., Spirohn K., Hao T., Calderwood M.A., Hill D.E., Landthaler M., Olivet J., Twizere J.C., Vidal M, & Wanker E.E. (2023). AI-guided pipeline for protein-protein interaction drug discovery identifies a SARS-CoV-2 inhibitor. bioRxiv.

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