Vp itc calorimeter

Manufactured by Malvern Panalytical

Sourced in United States, United Kingdom

The VP-ITC calorimeter is a high-sensitivity isothermal titration calorimeter designed for determining thermodynamic parameters of molecular interactions. It precisely measures the heat of interactions between two or more molecules, providing data on binding affinity, stoichiometry, and reaction enthalpy.

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VP-ITC (144 citations) MicroCal VP-ITC calorimeter (36 citations) VP-ITC titration calorimeter (14 citations) VP-ITC isothermal titration calorimeter (9 citations) MicroCal VP-ITC titration calorimeter (8 citations) MicroCal VP-ITC isothermal titration calorimeter (4 citations) VP-ITC 200 Microcal calorimeter (2 citations)

110 protocols using vp itc calorimeter

Thermodynamic Analysis of SLII-DMA Binding

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The RNA and the respective DMA samples were prepared and purified as described above. Calorimetric titrations were performed on a VP-ITC calorimeter (Microcal, LLC) at 25 °C into 10 mm K₂HPO₄, 20 mm KCl, 0.5 mm EDTA, and 4 mm BME (pH 6.5) buffer, centrifuged and degassed under vacuum before use. SLII at 40 μm was titrated into ~1.4 mL of 1.5 μm of the respective DMA over a series of 32 injections set at 6 μL each. To minimize the accumulation of experimental error associated with batch-to-batch variation, titrations were performed in triplicate. Data were analyzed using KinITC routines supplied with Affinimeter^{25 (link)}.
The AUF1 construct used in this study was prepared and purified as described above. Calorimetric titrations were performed on a VP-ITC calorimeter (Microcal, LLC) at 25 °C into 10 mm K₂HPO₄, 20 mm KCl, 0.5 mm EDTA, and 4 mm BME (pH 6.5) buffer, centrifuged and degassed under vacuum before use. AUF1 at 100 μm was titrated into ~1.4 mL of 8 μm SLII:DMA-135 complex at the following RNA:DMA ratios: 1:0, 1:1.6, 1:3.3, 1:5. To minimize the accumulation of experimental error associated with batch-to-batch variation, titrations were performed in duplicate. Data were analyzed using KinITC routines supplied with Affinimeter.

Davila-Calderon J., Patwardhan N.N., Chiu L.Y., Sugarman A., Cai Z., Penutmutchu S.R., Li M.L., Brewer G., Hargrove A.E, & Tolbert B.S. (2020). IRES-targeting small molecule inhibits enterovirus 71 replication via allosteric stabilization of a ternary complex. Nature Communications, 11, 4775.

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Calorimetric Analysis of Tp34 Binding to hLF

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Titrations were carried out in a VP-ITC calorimeter (Malvern Instruments, Malvern, UK). A typical titration consisted of 32 8-μL injections of wild-type or mutant Tp34 (408 – 455 μM) into a stirred reaction cell of about 1.4 mL containing hLF (18.0 – 18.5 μM). The reference power was 10 μcal/s, and the stirring rate was 307 rpm. All titrations were performed in triplicate with identical concentrations and injection schedules.

, & Brautigam C.A. (2014). Fitting two- and three-site binding models to isothermal titration calorimetric data. Methods (San Diego, Calif.), 76, 124-136.

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Calorimetric Analysis of UP1-RNA Interactions

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The UP1 construct used in these studies was prepared and purified as previously described [20 , 63 (link)]. Calorimetric titrations for ΔC_p were performed on a VP-ITC calorimeter (Malvern) at each temperature examined into 10 mM K₂HPO₄, 40 mM KCl, and 0.5 mM sodium ethylene-diaminetetraacetic acid (EDTA), pH 6.5. (His)₆-UP1 at 40μM was titrated into ~1.4mL of 1.0–1.5μM RNA over a series of 42 injections set at 6μL. To minimize the accumulation of experimental error associated with batch-to-batch variation, titrations were performed in duplicate at each temperature. Prior to non-linear least squares fitting in Origin v7.0, the raw data was corrected for dilution as previously described [20 ]. All data was fit to a two independent sets of sites binding isotherm.

Tolbert M., Morgan C.E., Pollum M., Crespo-Hernández C.E., Li M.L., Brewer G, & Tolbert B.S. (2017). HnRNP A1 Alters the Structure of a Conserved Enterovirus IRES Domain to Stimulate Viral Translation. Journal of molecular biology, 429(19), 2841-2858.

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Thermodynamic Analysis of Calmodulin-CDZ Interaction

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ITC experiments were performed using a VP-ITC calorimeter (Malvern Panalytical, Orsay, France). The ITC experiments were performed in buffer F at 25°C (unless otherwise stated). For a typical titration, the solution of analyte (8 μM of holo-CaM) was loaded in the reaction cell. The titrant (200 μM of CDZ) was loaded into the syringe before injection of 5 to 15 μl of the titrant into the reaction cell at intervals of 600 s. Heats of dilutions were measured by injecting the titrant into the buffer and were subtracted from the heat of reaction. The titration profiles were analyzed using the Origin7 software (OriginLab, Northampton, MA, USA) to determine the thermodynamic parameters, as previously described [35 (link)].

Léger C., Pitard I., Sadi M., Carvalho N., Brier S., Mechaly A., Raoux-Barbot D., Davi M., Hoos S., Weber P., Vachette P., Durand D., Haouz A., Guijarro J.I., Ladant D, & Chenal A. (2022). Dynamics and structural changes of calmodulin upon interaction with the antagonist calmidazolium. BMC Biology, 20, 176.

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Thermodynamic Characterization of RNF111-UBXN7 Binding

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ITC investigation was done using a VP-ITC calorimeter (Malvern Panalytical) as described in [29 (link)]. Purified proteins were dialyzed into a single stock of phosphate-buffered saline, pH 7,4 containing 1 mM Tris (2-carboxyethyl) phosphine hydrochloride (TCEP). All solutions were degassed at room temperature immediately prior to use in the test. Following thermal equilibrium at 25 °C, 10 µl of RNF111 RING domain at 200 µM was injected 20 times every 3.5 min into 10 µM of UBXN7-UAS domain or UBXN7-WT, or into 15 µM of UBXN7-∆UAS. The heat associated with each titration peak was integrated and plotted against the respective molar ratio of titrant and the different ligands. To correct for heats of dilution from the titrant, control experiments were performed by making identical injections of the titrant solution into a cell containing only the buffer and these heat effects were integrated with Origin 7.0 software (OriginLab). Binding stoichiometries and K_d values were determined by fitting corrected data to a single binding site model.

Amhaz S., Boëda B., Chouchène M., Colasse S., Dingli F., Loew D., Henri J., Prunier C, & Levy L. (2023). The UAS thioredoxin-like domain of UBXN7 regulates E3 ubiquitin ligase activity of RNF111/Arkadia. BMC Biology, 21, 73.

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Investigating Protein-Ligand Interactions

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ITC experiments were performed with a VP-ITC calorimeter (Malvern, MicroCal, Inc.), having 1.45-ml volume sample cell and a 278-μl volume syringe. The protein samples (wildtype AcrB, AcrB-I27A, AcrB-N298A, AcrB-V340A) were purified in a buffer containing 20 mM Tris-Cl, pH 7.5, 10 mM NaCl and 0.02% DDM. The syringe solution was also prepared in the same buffer and the total ionic strength was adjusted to 10 mM with NaCl. The titrations were performed under following conditions: wildtype AcrB or AcrB variant (3.5 μM) was titrated with 4 mM FUA in the syringe. The first injection was 3 μl followed by 54 further injections of 5 μl each. All titrations were performed at 25°C. The time interval between two injections was 180 s and the stirring speed was set to 307 rpm. All measurements were performed with high gain mode. Data was evaluated by using MicroCal Origin software provided by the manufacturer. The one site model was used for curve fitting. The first injection was omitted during data processing because of inaccuracy in volume associated with it. This first data point was therefore removed before analysis.

Tam H.K., Malviya V.N., Foong W.E., Herrmann A., Malloci G., Ruggerone P., Vargiu A.V, & Pos K.M. (2019). Binding and transport of carboxylated drugs by the multidrug transporter AcrB. Journal of molecular biology, 432(4), 861-877.

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Quantifying β-catenin-TCF7L2 Interactions

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ITC measurements were performed using the VP-ITC calorimeter (Malvern Pananalytical, Malvern, UK). Both β-catenin and TCF7L2 constructs were stored as lyophilised powders after labelling. To ensure matched buffers, lyophilised β-catenin was resuspended in ultra-pure water, the buffer was exchanged into PBS, 1 mM DTT, and the TCF7L2 was resuspended in the same buffer. Unlabelled TCF7L2 was washed with acetone three times, as previously described, to produce a lyophilised pellet. β-catenin was used at a concentration of 2–4 µM in the cell, and TCF7L2 was used at 6–100-fold higher concentration in the syringe (18–40 µM). Titrations of TCF7L2 into the buffer and the buffer into β-catenin were performed as controls. All experiments were performed at 30 °C. Data were fitted the Origin software package supplied with the instrument using the one-site binding equation. Data is shown in Supplementary Materials.

Smith B.M., Rowling P.J., Dobson C.M, & Itzhaki L.S. (2021). Parallel and Sequential Pathways of Molecular Recognition of a Tandem-Repeat Protein and Its Intrinsically Disordered Binding Partner. Biomolecules, 11(6), 827.

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Thermodynamic Analysis of ComR Protein Binding to XIP Peptides

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Purified ComR S. suis and variant proteins (K260A, N220A, and Q40A) were dialyzed overnight at 4°C into the same buffer stock of 20 mM Tris pH 7.5 100 mM NaCl 1 mM β-ME. HPLC purified synthetic peptides for both S. suis 05ZYH33 (WGTWVEE) and S. mutans U159 (GLDWWSL) were obtained from NeoScientific (Woburn, MA). Each XIP was reconstituted in the experimental buffer followed by centrifugation at 12,000 rpm to clear undissolved or precipitating material. XIP was used at 300 μM concentration and injected into 20 μM ComR. All experiments were performed using a VP-ITC calorimeter (Malvern) at 25°C. Controls included titration of XIP into buffer alone or the titration of buffer into ComR. The final heats of binding were analyzed using Origin Software (Malvern) using a one-site model.

Shanker E., Morrison D.A., Talagas A., Nessler S., Federle M.J, & Prehna G. (2016). Pheromone Recognition and Selectivity by ComR Proteins among Streptococcus Species. PLoS Pathogens, 12(12), e1005979.

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Isothermal Titration Calorimetry of Protein-Ligand Interactions

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Purified GluN1a–GluN2B ATD proteins from the above-described fusion protein purification was dialyzed extensively against a buffer containing 150 mM NaCl, 20 mM sodium phosphate, and 5% glycerol. The final pH of the solution was achieved using a ratio of dibasic:monobasic sodium phosphate for pH values 6.5 and 7.6.
After dialysis, the protein sample was diluted to 10–15 μM and dimethyl sulfoxide (DMSO) was added to a final concentration of 1.5% (v/v), in order to match that of the ligand solution. Ligands were dissolved in DMSO to a stock concentration of 10 mM, and then further diluted to 150–200 μM using buffer from the protein dialysis. The protein and ligand solutions were thoroughly degassed, and the protein solution was placed in a 1.4 mL cell of a VP ITC calorimeter (Malvern) with the experimental temperature set to 30 °C and differential power at 15 μCal s⁻¹. The respective ligands were injected at 300 s intervals after an initial delay of 180 s. Dilution enthalpy was calculated from post-saturation injections and subtracted from total heats, and the data were fit using a single-site model with the program OriginLab 7.5.

Regan M.C., Zhu Z., Yuan H., Myers S.J., Menaldino D.S., Tahirovic Y.A., Liotta D.C., Traynelis S.F, & Furukawa H. (2019). Structural elements of a pH-sensitive inhibitor binding site in NMDA receptors. Nature Communications, 10, 321.

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Measuring Protein-Protein Interactions by ITC

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ITC measurements were carried out on a Microcal VP-ITC calorimeter (Malvern) at 25 °C. Proteins used for ITC measurements were dissolved in an assay buffer composed of 50 mM Tris, 100 mM NaCl, 1 mM EDTA, and 1 mM DTT at pH 7.8. High concentrations of proteins (e.g., 200 μM for Trx-E-cadherin) were individually loaded into the syringe and titrated into the cell containing low concentrations of the corresponding proteins (e.g., 20 μM for Trx-AnkG R1-24). For each titration point, a 10 μl aliquot of a protein sample in the syringe was injected into the protein in the cell at a time interval of 120-80 seconds. ITC titration data were analyzed using Origin7.0 software and fitted by the one-site binding model. The n values for all ITC experiments are listed in the figure or summary table.

Kong C., Qu X., Liu M., Xu W., Chen D., Zhang Y., Zhang S., Zhu F., Liu Z., Li J., Huang C, & Wang C. (2023). Dynamic interactions between E-cadherin and Ankyrin-G mediate epithelial cell polarity maintenance. Nature Communications, 14, 6860.

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