Microscale thermophoresis

Manufactured by NanoTemper

Sourced in Germany

Microscale thermophoresis is a laboratory technique that measures the movement of molecules in a temperature gradient. It provides information about molecular interactions, binding affinities, and sample quality.

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Lab products found in correlation

Red tris nta dye, by NanoTemper (1 mentions) Mo affinity analysis v2, by NanoTemper (1 mentions) Prism 8, by GraphPad (1 mentions)

5 protocols using microscale thermophoresis

Determination of PFKFB3 Binding Affinity

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The dissociation constant was determined using Nanotemper microscale thermophoresis (MST)^{38 (link)}. In a typical experiment, a compound was dissolved in MST buffer and mixed with 100 nM RED-NHS labeled recombinant PFKFB3 in 1:1 ratio. The mixture was incubated at room temperature for 20 min. and a full MST measurement was performed. Data were analyzed with Nanotemper software (https://nanotempertech.com/monolith/).

Macut H., Hu X., Tarantino D., Gilardoni E., Clerici F., Regazzoni L., Contini A., Pellegrino S, & Luisa Gelmi M. (2019). Tuning PFKFB3 Bisphosphatase Activity Through Allosteric Interference. Scientific Reports, 9, 20333.

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Monobody Binding to EmrE Quantified

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Monobody L10 was labeled at a unique, introduced cysteine, A13C, with fluorescein maleimide. Binding to EmrE₃ was measured using microscale thermophoresis (Nanotemper, Munich, Germany). For these experiments, labeled monobody was held constant at 2 μM, and the concentration of EmrE₃ was varied from 30 nM to 100 μM. Buffer contained 100 mM NaCl, 10 mM HEPES, pH 7, 4 mM DM, and 50 μg/mL bovine serum albumin. Samples were incubated at least 30 min prior to measurement of binding interactions. Experiments were performed using three independent sample preparations and fit to a one site binding equilibrium with total L10 as the experimental variable:

M S T ([E m r E]) = {M S T}_{0} + \frac{({M S T}_{f} - {M S T}_{0})}{2} (1 + \frac{[E m r E]}{[L 10]} + \frac{K_{D}}{[L 10]}) [1 - \sqrt{1 - \frac{4 \frac{[E m r E]}{[L 10]}}{{(1 + \frac{[E m r E]}{[L 10]} + \frac{K_{D}}{[L 10]})}^{2}}}]

where MST([EmrE]) is the MST signal as a function of total EmrE added to a fixed concentration of labelled L10 monobody, and MST₀ and MST_f are the arbitrary initial and final MST fluorescence signals.

Kermani A.A., Burata O.E., Koff B.B., Koide A., Koide S, & Stockbridge R.B. (2022). Crystal structures of bacterial small multidrug resistance transporter EmrE in complex with structurally diverse substrates. eLife, 11, e76766.

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Quantifying NorC-TPP+ Binding Dynamics

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Quantification of NorC’s interaction with TPP⁺ was carried out using microscale thermophoresis (Nanotemper)^{56 (link)}. The protein was labeled with red Tris-NTA dye (Nanotemper) by mixing both in an equimolar proportion. The labeled protein was mixed with TPP⁺ such that the concentration of the labeled protein and TPP⁺ were 10 nM and 0.1 mM, respectively. A total of 16 twofold serial dilutions of TPP⁺ were prepared to keep the protein concentration constant at 10 nM. The experiment was carried out with Monolith™ NT.115 MST premium-coated capillaries. For NorC-ICab complex titrations against TPP⁺, 10 nM of NorC was mixed with ICab in a 1:3 molar ratio and the starting concentration of TPP⁺ was kept at 0.1 mM. For ICab titrations against NorC, 10 nM of NorC was used with a starting concentration of 5 µM for the ICab. The curves were fitted with the single-site binding model. To assess the same with NorB and ICab, identical concentrations were taken, with NorB in place of NorC.

Kumar S., Athreya A., Gulati A., Nair R.M., Mahendran I., Ranjan R, & Penmatsa A. (2021). Structural basis of inhibition of a transporter from Staphylococcus aureus, NorC, through a single-domain camelid antibody. Communications Biology, 4, 836.

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Evaluation of Compounds for PknB Activity

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Example 2

Compounds were tested for PknB activity either using the ATP Glo® assay as described or by direct binding using microscale thermophoresis (NanoTemper Technologies) (FIG. 2). Compounds were also tested microbiologically against an auxotrophic M. tuberculosis following published procedures. The compounds displayed a surprising range of biochemical and microbiological activity, though 14 out of 20 were microbiologically better than GW779439X (FIG. 2, FIGS. 4a and 4b, FIG. 6 (PknB inhibition by ATP competition assay), FIG. 7 (PknB physical binding), FIG. 8 (M. tuberculosis auxotroph activity without beta-lactam antibiotic), FIG. 9 (Cdk2 inhibition by ATP competition assay), FIG. 10 (Toxicity in zebra fish), and FIG. 11 (Toxicity in mouse primary macrophage cells)). It is unclear whether this is due to off target effects on one of the other 10 serine threonine kinases in M. tuberculosis or improved cellular accumulation, but this is difficult to test since PknB is essential and a genetic knockout is not viable. Three Boc-protected intermediates were tested (UW130 shown) and all showed no appreciable activity toward the kinase or bacteria, suggesting a functional role for the protonated methylpiperidine. These data show the ability to create novel compounds in this class with potential for improved microbiological activity and possible activity against other kinases.

US11820753B2. Inhibitors of bacterial pasta kinases (2023-11-21). Wisconsin Alumni Research Foundation [US]. Inventors: Robert Todd Striker [US], Nathan Joseph Wlodarchak [US], John Bruce Feltenberger [US], Jennifer Golden [US].

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Measuring PBP3 Binding Affinity

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The affinity of 1 and 33a with PBP3 was determined by microscale thermophoresis (NanoTemper Technologies GmbH, Germany) according to the manufacturer's instructions. MO. Affinity Analysis v2.1.3 software (NanoTemper Technologies GmbH, Germany) was used to analyze the data, and the fitted curve was obtained by GraphPad Prism 8 (GraphPad Software, USA).

Li Z., Guo Z., Lu X., Ma X., Wang X., Zhang R., Hu X., Wang Y., Pang J., Fan T., Liu Y., Tang S., Fu H., Zhang J., Li Y., You X, & Song D. (2023). Evolution and development of potent monobactam sulfonate candidate IMBZ18g as a dual inhibitor against MDR Gram-negative bacteria producing ESBLs. Acta Pharmaceutica Sinica. B, 13(7), 3067-3079.

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