All experiments were conducted on a VP microcalorimeter (Microcal, Amherst, MA, USA) at 25 °C. For the analysis of the CheB homologs of P. aeruginosa, 15–40 μM of proteins (dialyzed into 5-mM Tris/HCl, 5-mM Pipes, and 5-mM Mes, pH 7.0) were placed into the sample cell and titrated with 1–7-mM solution of the GWEEF peptide (synthesized by Biomedal S.L., Seville, Spain). For the analysis of CheB_Pec, the protein was dialyzed into 5-mM Tris/HCl, 5-mM PIPES, 5-mM MES, 10% (v/v) glycerol, 2-mM dithiothreitol, 150-mM NaCl, and 0.1-mM EDTA, pH 7.4, adjusted to 15–50 μM and titrated with 4.8–14.4-μL aliquots of 1–5-mM peptide solutions (synthesized by GenScript®, Piscataway, NJ, USA). All ligand solutions were prepared in dialysis buffer immediately before use. The mean enthalpies measured from the injection of the peptide into the buffer were subtracted from the raw titration data prior to data analysis with the “one binding site model” of the MicroCal version of ORIGIN.
Vp microcalorimeter
Manufactured by Malvern Panalytical
Sourced in United States
The VP-microcalorimeter is a high-sensitivity instrument designed for the measurement of heat effects associated with chemical and biological processes. It measures the heat generated or absorbed during a reaction or process, providing quantitative data on the energetics of these events.
Automatically generated - may contain errors
Lab products found in correlation
18 protocols using vp microcalorimeter
1
Biophysical Characterization of CheB Homologs
2
Calorimetric Analysis of PP4888-LBD Binding
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Experiments were conducted on a VP-microcalorimeter (Microcal, Amherst, MA, United States) at 15°C. PP4888-LBD was dialyzed into 20 mM Hepes, 150 mM NaCl, 10% (v/v) glycerol, pH 7.4, adjusted to 50 μM and titrated with 0.5 mM of DIBOA prepared in dialysis buffer.
3
Protein-Ligand Binding Affinity Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Experiments were conducted on a VP‐microcalorimeter (Microcal). Proteins were dialyzed into the TMP buffer, placed at a concentration of 20–40 µM into the sample cell and titrated with 0.25–2.5 mM ligand solutions that were prepared using the dialysis buffer immediately before use. In the case no binding heats were observed in titrations with 14.42 μL aliquots of 10–20 mM ligand solution, it was concluded that there was no binding. The mean enthalpies measured from the injection of ligands into the buffer were subtracted from raw titration data before data analysis with the MicroCal version of ORIGIN. Data were fitted with the “One binding site model” of ORIGIN.
4
Microcalorimetric Titration of GltS-LBD
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Microcalorimetric titrations were carried out at 25°C using a VP-microcalorimeter (Microcal, Amherst, Massachusetts, USA). GltS-LBD was dialyzed against 50-mM Hepes, pH 7.9, 300-mM NaCl, 1-mM dithiothreitol and 10% (v/v) glycerol and ligand solutions were made up in dialysis buffer. The titration involved 3.2-μM injections of 1–20-mM effector solutions into 20-μM GtrS-LBD. Control experiments involved the injection of effector solution into dialysis buffer. Raw titration data were concentration-normalized and corrected for dilution effects prior to analysis using the ‘One-binding site model’ of the MicroCal version of ORIGIN. The parameters ΔH (reaction enthalpy), KA (binding constant, KA = 1/KD) and n (reaction stoichiometry) were determined from the curve fit. The changes in free energy (ΔG) and entropy (ΔS) were calculated from the values of KA and ΔH with the equation: ΔG = −RT ln KA = ΔH − TΔS, where R is the universal molar gas constant and T is the absolute temperature (23 (link)).
5
Isothermal Titration Calorimetry of Protein-Ligand Interactions
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Experiments were performed on a VP microcalorimeter (Microcal, Amherst, MA, USA) at 25°C. Proteins were placed into the sample cell (36 to 65 µM). Compound solutions (1 to 5 mM) were prepared in dialysis buffer and placed into the injector syringe. Titrations involved the injection of 9.6-µl to 19.2-µl aliquots of compound solution into the protein. In cases in which no binding was observed, the experiment was repeated at an analysis temperature of 15°C. The mean enthalpy values from the titration of buffer with compounds were subtracted from raw titration data prior to data analysis performed with the “One binding site model” of the MicroCal version of ORIGIN.
6
Microcalorimetric Phosphatase Activity Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Microcalorimetric phosphatase activity assays were conducted at 25°C using a VP‐microcalorimeter (Microcal, Amherst, Massachusetts, USA; Todd & Gomez, 2001 (link); Bianconi, 2003 (link); Krell, 2008 (link), Zambelli, 2019 ). The purified phosphatase and substrate solutions were dialysed against a buffer made of 40 mM MES, 40 mM HEPES, 40 mM acetic acid, 150 mM NaCl, 1 mM DTT, and 10% [v/v] glycerol at pH 4, 5.5, 7, and 8.5. Substrates were prepared in the same buffer. The titration involved injecting 10 μL of 500 μM substrate solution into 0.1–1 μM of purified FS6 acid phosphatase in the same buffer. Control experiments consisted of injecting the substrate solution into the dialysis buffer. Raw titration data were normalized for concentration and corrected for dilution effects before analysis using the ‘Enzyme kinetics—single injection model’ of the MicroCal PEAQ‐ITC analysis software. The parameter ΔH (reaction enthalpy) was determined through curve fitting (Abadou & Labdury, 2006 (link)).
7
Thermodynamics of Protein-Ligand Binding
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Freshly purified proteins were dialyzed into the buffers specified in Table S1 and introduced into the sample cell of an VP-microcalorimeter (MicroCal). Proteins at 15 to 100 μM were titrated with 0.25 to 10 mM ligand solutions that were made up in dialysis buffer. For pH-active ligands, the pH of the ligand solutions was adjusted to that of the dialysis buffer by the addition of concentrated HCl or NaOH. Titrations of MBP and McpV-LBD were conducted at 15°C to favor endothermic contributions to binding heats, whereas the remaining titrations were carried out at 25°C. Raw titration data were integrated, corrected for ligand-dilution heats and normalized with the ligand concentrations. Resulting data were fitted with the “One Binding site model” of the MicroCal version of ORIGIN. Provided are the dissociation constants derived from one experiment and the error of curve-fitting.
8
Quantifying Protein-Ligand Interactions
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Experiments were conducted on a VP-microcalorimeter (Microcal, Amherst, MA, USA) at 20°C or 10°C. McpK-LBD was dialyzed overnight against 5 mM Tris, 5 mM PIPES, 5 mM MES, pH 8.0 and placed into the sample cell. Typically, protein at 20–100 μM was introduced into the sample cell and titrated with 1–3 mM ligand solutions that were prepared in dialysis buffer immediately before use. For fitting, data were integrated using NITPIC (Keller et al., 2012 (link)) before global fitting to a two symmetric-site binding model in SEDPHAT (Houtman et al., 2007 (link)). The binding constants expressed are corrected as Kd1 = 2∗Kd1′ and Kd2 = 0.5∗Kd2′ in order to express an estimation of the microscopic constants for the two binding sites model, where Kx’ are the macroscopic constants measured. The cooperativity factor α is expressed as α = Kd1/Kd2. Statistical uncertainties for best-fit estimates of Kd and ΔH were calculated using standard error surface projection methods built into SEDPHAT.
9
Protein-Ligand Binding Thermodynamics
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Experiments were conducted on a VP-microcalorimeter (Microcal, Amherst, MA, USA) at a temperature of 25 °C. Freshly purified protein, at 10 to 169 µM, was dialysed into analysis buffer, placed into the sample cell of the instrument and titrated with ligand solutions at 200 µM to 5 mM. Typically, a single injection of 1.6 µL was followed by a series of 4.8 or 6.4 µL aliquots. The mean enthalpies measured from the injection of ligand solutions into the buffer were subtracted from raw titration data. Data were normalized with the ligand concentrations, the first data point removed and the remaining data fitted with the ‘One Binding Site’ model of the MicroCal version of ORIGIN (Microcal, Amherst, MA, USA).
10
Calorimetric Analysis of PctD-LBD Mutants
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Experiments were conducted on a VP-microcalorimeter (Microcal, Amherst, MA). The site-directed mutants of PctD-LBD were analyzed as reported previously (17 (link)). In the case no binding heats were observed, experiments were repeated with a higher choline concentration and the resulting curves (Fig. S1 ) were used to derive the dissociation constants (Table 1 ). Proteins R1 to R10 were dialyzed into the analysis buffer, placed at a concentration of 10 to 75 µM into the sample cell and titrated with freshly made up amine solutions (0.5 to 20 mM). In the case no binding heats were observed for a titration with 14.42 μL aliquots of 10–20 mM ligand solution, it was concluded that there was no binding. The mean enthalpies measured from the injection of effectors into the buffer were subtracted from raw titration data prior to data analysis with the MicroCal version of ORIGIN. Data were fitted with the ‘One binding site model’ of ORIGIN.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!