Cl– binding to the A399C/A432C mutant of CLC–ec1 was carried out as described36 (link),38 (link) using a nanoITC instrument (TA Instruments). For these experiments, the final purification step of the protein was purified over a gel filtration column pre-equilibrated in 100 mM Na-K-Tartrate, 20 mM Hepes, 50 μM DMNG, pH 7.5 (Buffer B0) and concentrated to 50-195 μM. The A399C/A432C mutant was not stable in the absence of Cl– upon incubation with Hg2+. Therefore, in this case the Hg2+ reaction was carried out in Buffer B. Excess Cl– and Hg2+ were successively removed by running the protein over a gel filtration column pre-equilibrated in buffer B0. The injection syringe was filled with buffer B0 with 50 mM KCl added, to achieve final Molar Ratios of 100-250. Each experiment consisted of 30-48 injections of 1 μl of the ligand solution at 3-4 min intervals into the experimental chamber kept under constant stirring at 350 rpm and at 25.0±0.1 °C. All solutions were filtered and degassed prior to use. The ITC data was fit to a single site Wiseman isotherm as described36 (link),38 (link) using the NanoAnalyze program from TA instruments.
Nanoanalyze program
Manufactured by TA Instruments
NanoAnalyze is a software program developed by TA Instruments to provide data analysis and reporting capabilities for their line of nanoscale characterization instruments. The program is designed to process and analyze data collected from these instruments, allowing users to visualize and interpret their experimental results.
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8 protocols using nanoanalyze program
1
Chloride Binding to CLC-ec1 Mutant
2
Chloride Binding to CLC-ec1 Mutant
3
PDZD8-Rab7 Interaction Quantified by ITC
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The protein–protein interaction of the PDZD8 CC and Rab7 was analyzed by isothermal titration calorimetry (ITC) using an Affinity ITC calorimeter (low volume cell 190 μl; TA instruments) at 20 °C. The PDZD8 CC constructs used for the ITC experiments were fused to the His-tagged maltose-binding protein (MBP) in their N-termini for protein stability. The purified Rab7 Q67L and Rab7 T22N were loaded with 1 mM of GTP and GDP, respectively. All proteins were prepared in the identical buffer containing 20 mM Tris–HCl pH 7.5, 150 mM NaCl. The syringe was loaded with 1 mM of the MBP-PDZD8 CC and the cell was filled with 300 μl of 0.1 mM Rab7. The titration curve was obtained by injecting 2 μl × 25 aliquots of the MBP-PDZD8 CC into the cell at a time interval of 300 s. The enthalpy of reaction, ΔH0, the binding constant, Kd, and the stoichiometry value, n, were calculated from the measured heat changes, δHi, upon the association of the MBP-PDZD8 CC and Rab7. The titration data were analyzed using the NanoAnalyze program (TA instruments) and fitted into a two-site binding model for wild type and an independent binding model for mutants.
4
Binding Affinity Measurements of MgCl2 and AMPPNP
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The binding affinity measurements for MgCl2 and AMPPNP and the indicated protein were taken with a Nano-ITC (TA Instruments). Experiments were carried out at 25 °C with 0.03 mM of protein in the cell and mixed with 0.7 or 5 mM nucleotide in the syringe by 25 injections of 2 μl each at intervals of 180s under continuous stirring. Data integration, correction and analysis were carried out using NanoAnalyze program (TA Instruments) with a single-site binding model.
5
Measuring Chloride Binding Affinity of CLC-ec1 F357A
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Cl- binding affinity was determined for purified CLC-ec1 F357A as described (Picollo et al., 2009 (link); Basilio et al., 2014 (link)) using a nanoITC instrument (TA Instruments). For these experiments, the final purification step of the protein was purified over a gel filtration column pre-equilibrated in 100 mM Na-K-Tartrate, 20 mM Hepes, 50 μM DMNG, pH 7.5 (Buffer B0) and concentrated to 50–195 μM. The injection syringe was filled with buffer B0 with 50 mM KCl added, to achieve final Molar Ratios of 70–100. Each experiment consisted of 30–48 injections of 1 μl of the ligand solution at 3–4 min intervals into the experimental chamber kept under constant stirring at 350 rpm and at 25.0 ± 0.1°C. All solutions were filtered and degassed prior to use. The ITC data was fit to a single site Wiseman isotherm using the NanoAnalyze program from TA instruments.
6
Binding Affinity of NmMafB2-CT2 and NmMafI2
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The affinity between NmMafB2-CT2 and NmMafI2 was measured at 20 °C using a Nano ITC calorimeter (low volume cell 190 μl; TA instruments). All samples were prepared in the same buffer (10 mM Tris–HCl pH 8.0, 100 mM NaCl and 2 mM β-ME). Titration was performed by injecting 1 μl of NmMafI2 (~ 1.3 mM) into the cell containing NmMafB2-CT2 (~ 0.2 mM) 50 times at 250 s intervals. The NanoAnalyze program (TA Instruments) was used to calculate the interaction parameters after fitting the data with the independent binding sites model. The titration curve used for the program analysis was baseline-corrected with the buffer used in the titration.
7
Isothermal Titration Calorimetry of wAlb12-Co(II) Interaction
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The wAlb12–Co(II) interaction was analyzed by isothermal titration calorimetry (ITC) using an Affinity ITC calorimeter (TA instruments, New Castle, DE, USA) at 25 °C. All of the samples were prepared in the identical buffer containing 50 mM sodium phosphate with 10 mM NaCl. The syringe was loaded with 5 mM of the CoCl2 and the cell was filled with 350 μL of 0.5 mM wAlb12. The titration curve was obtained by injecting 2 μL × 25 aliquots of the CoCl2 into the cell at a time interval of 300 s. The enthalpy of the reaction, ΔH0, the dissociation constant, Kd, and the stoichiometry value, n, were calculated from the measured heat changes, δHi, upon the association of the wAlb12 and CoCl2. The titration data were analyzed using the NanoAnalyze program (TA instruments) and fitted into a single-site binding model.
8
Cdc5-Dbf4 and Cdc5-Spc72 Interactions Analysis
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The Cdc5-Spc72P and Cdc5-Dbf4 interactions were analyzed using the Nano ITC instrument (TA Instruments), while the interaction between Cdc5-A567W and Dbf4 was analyzed using the MicroCaliTC200 instrument (Malvern Instruments Inc.). Peptides derived from Dbf4 (73EKKRARIERARSIEGAVQVSKGTG96), and Spc72P (222DKEEFLSLAQSpS PAGSQ237), as well as a non-phosphorylated variant of the Spc72 peptide were purchased from GenScript and resuspended in storage buffer supplemented with 20 mM EDTA pH 8.0. The Cdc5-peptide interactions analyzed using the Nano ITC instrument were designed by filling the chamber cell with 30 µM protein and the injection syringe with 280 µM peptide. The ternary complex (Dbf4-Cdc5-Spc72P) was analyzed by filling the chamber cell with either Cdc5-Dbf4 or Cdc5-Spc72P (at a 1:4 molar ratio of protein:peptide), and the injection syringe with 280 µM peptide. Data was processed using the NanoAnalyze program (TA Instruments). The interaction between Cdc5-A567W and Dbf4 was analyzed with 45 µM protein in the chamber cell and 1.2 mM peptide in the injection syringe. Data was processed using the MicroCaliTC200 program (Malvern Instruments Inc.).
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