PCNA and the TRAIP447–469 peptide were extensively dialyzed into 25-mM Hepes, pH 7.4, 150-mM NaCl, and 0.25-mM tris(2-carboxyethyl)phosphine. Both protein and peptide concentrations were determined using UV spectroscopy and molar extinction coefficients at 280 nm (see the previous section). ITC experiments were performed at 25°C using a calorimeter (MicroCal Auto-iTC200; Malvern). The ITC experiments used an initial delay of 120 s and were divided into 25 injections of 1.5 µl. Control experiments of peptide into buffer were performed to measure heat dilution effects, which were found to be negligible. The experimental binding isotherms were fitted by nonlinear least squares fitting to a model assuming a single set of equivalent sites using software provided by the MicroCal Auto-iTC200 manufacturer.
Microcal auto itc200
Manufactured by Malvern Panalytical
Sourced in United Kingdom, United States
The MicroCal Auto-iTC200 is a calorimetry instrument designed for the automated measurement of thermodynamic properties of molecular interactions. It provides high-throughput analysis of biomolecular interactions through its advanced sample handling and data analysis capabilities.
Automatically generated - may contain errors
Lab products found in correlation
Origin software, by OriginLab (3 mentions)
Microcal itc software v1, by Malvern Panalytical (2 mentions)
Microcal peaq itc analysis software, by Malvern Panalytical (2 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (2 mentions)
Origin 7, by Malvern Panalytical (2 mentions)
Microcal itc200, by Malvern Panalytical (2 mentions)
Origin 7, by OriginLab (2 mentions)
Itc200, by Malvern Panalytical (1 mentions)
Sodium succinate, by Merck Group (1 mentions)
Sodium oxaloacetate, by Merck Group (1 mentions)
Malic acid, by Merck Group (1 mentions)
Cis aconitic acid, by Merck Group (1 mentions)
3 phenylpropionic acid, by Merck Group (1 mentions)
Itaconic acid, by Merck Group (1 mentions)
Lps e coli serotype 055 b5, by Merck Group (1 mentions)
Lps e coli o111 b4, by Merck Group (1 mentions)
Microcal origin 7, by Malvern Panalytical (1 mentions)
E coli o111 b4, by Merck Group (1 mentions)
Pi 4 5 p2 dic8, by Echelon Biosciences (1 mentions)
Na2so3, by Merck Group (1 mentions)
50 protocols using microcal auto itc200
1
Characterizing PCNA-TRAIP Peptide Binding
2
Isothermal Titration Calorimetry of Protein-Peptide Interactions
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Titrations were performed using the MicroCal iTC200 and MicroCal Auto-iTC200 instruments (Malvern Panalytical, Malvern, UK). Binding assays were carried out in 150 mM NaCl, 50 mM Tris pH 8, 1 mM DTT and 0–1% v/v DMSO. Generally, 13–20 injections were carried out with a DP of 6 μcal/s, 750 RPM, 20–25 °C, 150–180 s spacing and 5–6 s injection times of 2.5–3 μL. Titrations were performed with protein and peptide in cell and syringe interchangeably. Generally, TRIM7 in the cell was kept at 20 μM with peptides diluted to 400 μM and up to 2 mM for dipeptides. See Table S2 for details. Control titrations of titrant into buffer were carried out where appropriate. Data were analyzed using MicroCal PEAQ-ITC analysis software, using one site model to fit the data. For low c-value experiments (such as the LQ titration), the N was fixed to 1. For experiments with MBP-T7CCPS, the buffer was 165 mM NaCl, 50 mM Tris pH 8, 1 mM DTT, 0.5% w/v glycerol and 0–0.8% v/v DMSO. For experiments with rbGYG, mGYG and mTrim7-PRYPSRY, the protein samples were dialysed against 50 mM phosphate buffer, pH 6.5 and 1 mM DTT. ITC experiments were conducted on MicroCal ITC 200 at 15 °C or an AutoITC and analysed using a standard one-state model within MicroCal instrument software.
3
Calorimetric Binding Assay of Survivin-Sgo1
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ITC experiments were performed using a MicroCal Auto-iTC200 (Malvern Instruments). A total of 40 μl of 50–375 μM (monomer concentration) Survivin/CPC complexes was injected into 200 μl of 5–20 μM (monomer concentration) hSgo1 constructs in 16 aliquots (1 × 0.5 μl and 15 × 2.5 μl), 180 s between injections, reference power 3 µcal/s−1, syringe spin 750 rpm, and filter period 5 s. Control titrations were performed in which the injectant was added to buffer without protein or buffer was injected into the protein. Titrations were carried out at 20°C, except for the analysis of the Survivin/Sgo1AKER interaction, which was performed at 10°C. The heat of reaction was corrected for the heat of dilution and analyzed using the MicroCal ITC software v1.30 (Malvern Instruments). All experiments were carried out in 50 mM Hepes, pH 8, 150 mM NaCl, 5% (vol/vol) glycerol, and 1 mM tris(2-carboxyethyl)phosphine (TCEP), except the experiment to assess the binding affinity between CPC10–280 and Sgo11–415 or Sgo11–415 4A (Fig. S4, D and E ) that was carried out at 50 mM Hepes, pH 8, 250 mM NaCl, 5% (vol/vol) glycerol, 1 mM TCEP, and 0.005% Tween.
4
Quantifying LRP6-VHH Interactions
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Isothermal titration calorimetry (ITC) was performed using a MicroCal Auto ITC200 (Malvern Instruments Ltd). A 10 µM solution of LRP6P3E3P4E4 in DPBS was placed into the 200 μL sample cell at 25 °C. Titration was performed with 2 µL injections (first injection 0.5 µL) of VHHs in DPBS at a concentration of 100 µM every 120 s (26 injections total). Data were fitted using the MicroCal PEAQ-ITC Analysis Software (Malvern Instruments Ltd) according to standard procedures. Fitted data yielded the stoichiometry (n), the dissociation constant (kD), enthalpy (H) and entropy (S). Each ligand test was performed in triplicate and values for n, kD, H and S represent mean ± s.d. of three independent experiments.
5
Energetics of Gd3+ Binding to TRPV6
6
Isothermal Titration Calorimetry of RipR RD
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MicroCal Auto ITC200 (Malvern Panalytical) at the Korea Basic Science Institute was used for the ITC experiments. All samples were prepared in a buffer containing 20 mM Hepes (pH 7.0), 300 mM sodium chloride, and 2 mM β-mercaptoethanol. The ligands, potassium threo-isocitrate, itaconic acid, sodium succinate, malic acid, sodium oxaloacetate, cis-aconitic acid, and 3-phenylpropionic acid were purchased from Sigma–Aldrich. RipR RD (30 μM) was prepared in the sample cell, and each ligand (300 μM) was loaded into a titrating syringe. The titrations were measured with 19 2-μl injections with 150-s spacing at 25 °C.
7
Thermodynamics of LPS Binding to Thanatin
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Microcalorimetric measurements of the binding of thanatin or divalent cations (Mg2+, Ca2+) to LPS were performed on a MicroCal Auto-ITC200 instrument (Malvern Instruments, Malvern, UK)35 (link). LPS (E. coli serotype 055:B5, Sigma, USA) was dissolved in 20 mM Tris-HCl (pH = 6.8) or 10 mM PBS (pH = 7.4), vortexed vigorously for 15 min, and sonicated for 15 min at 60 °C. The LPS solution was sonicated for 5 min prior to use. Thanatin was dissolved in Tris-HCl (pH = 6.8) and titrated into LPS in Tris-HCl (pH = 6.8). Divalent cations (Mg2+, Ca2+) were dissolved in PBS (pH = 7.4) and titrated into LPS in PBS (pH = 7.4). All samples were degassed for 10 min in a sonication bath before the experiments. These experiments were performed at 25 °C. The generated peaks were integrated using Origin 7.0 software. The errors for all the reported thermodynamic parameters were estimated through Monte Carlo simulation with the standard errors of three experiments.
8
Characterizing SH2 Domain-Peptide Interactions
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Synthesized phosphotyrosyl peptides (95% or greater purity by HPLC) were purchased from Toray Research Center. Based on a pilot experiment with different peptide lengths, we found that 8-mer was the minimal length to obtain reliable ITC and NMR titration results (not shown). Isothermal titration calorimetry (ITC) was performed in MicroCal Auto-iTC200 (Malvern) at 25 °C as described in the manufacturer’s instruction manual. The SH2 domain and peptide were dissolved in 25 mM HEPES, pH7, 100 mM NaCl at a final concentration of 40 μM or 400 μM, respectively. Titration was carried out by injecting the peptide solution into the reaction cell holding the SH2 domain containing solution every 150 seconds. The results were analyzed by the software ORIGIN 7 supplied by the manufacturer.
9
Orco-VUAA1 Binding Kinetics
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Samples of Orco and Orco-Fab complex were expressed and purified as described above, and concentrated to ~10 μM (monomer). A VUAA1 (Princeton Biomedical Research) stock was prepared in dimethylsulfoxide (DMSO; Sigma-Aldrich) at 100 mM and diluted to 0.5 mM in the same buffer as Orco. 0.5% (v/v) DMSO was added to Orco and buffer samples to match the amount of DMSO originating from the VUAA1 stock solution. ITC experiments were performed using a MicroCal Auto-iTC200 (Malvern) at 25 °C. Each experiment began with a single injection of 0.4 μL followed by 19 injections of 2 μl each (at 0.5 μL/s, 150 s apart) into a 0.2-mL Orco sample. The experiments were repeated using Orco samples obtained from independent purifications (biological replicates).
The raw heat evolutions were baseline-corrected and a single binding site model was fit to the integrated data using AFFINImeter (https://www.affinimeter.com ), excluding the first injection. The number of binding sites per monomer was fixed to be 1 and a dissociation constant (Kd), enthalpy of binding (ΔH) and heat of sample dilution (ΔQdil) were fit. Separate experiments injecting VUAA1 into buffer alone showed no significant heats of dilution and were not subtracted from the Orco data prior to fitting.
The raw heat evolutions were baseline-corrected and a single binding site model was fit to the integrated data using AFFINImeter (
10
Measuring Peptide-LPS Binding Affinity by ITC
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To measure the binding affinity of a peptide to LPS, ITC experiments were performed using a MicroCal AutoiTC200 (Malvern Panalytical, Malvern, UK) at the Korea Basic Science Institute (KBSI, Ochang, Republic of Korea). T. ni cecropin (0.1 mM) was injected into 370 μL of 25 μM LPS (E. coli O111:B4, Sigma-Aldrich) in Dulbecco’s phosphate-buffered saline (DPBS, pH 7.0; Welgene) at 2.5 s intervals for 98 s at 37 °C for 38 injections. LPS was pretreated with 15 min vortex, 5 min heating at 60 °C, followed by 5 min sonication. The data were analyzed for binding affinity using MicroCal Origin software (MicroCal Origin, Northhampton, MA, USA).
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