Isothermal titration calorimetry (ITC) measurements were performed at 25°C, using an iTC200 Microcalorimeter (MicroCal Inc.). Experiments included 20 injections of 2 μl of SAM (1 mM) into the sample cell containing 100 μM FTSJ1, which was purified from insect cells. The SAM and FTSJ1 were kept in the same buffer (20 mM Tris–HCl, pH 7.5, 500 mM NaCl, and 0.5 mM TCEP). SAM was titrated in the same buffer and was used as a control. Binding isotherms were fit by non‐linear regression using Origin Software version 7.0 (MicroCal Inc.). The ITC data were fitted to a one‐site binding model using Origin Software version 7.0 (MicroCal Inc.).
Origin software version 7
Manufactured by Malvern Panalytical
Sourced in United States, United Kingdom
Origin Software version 7.0 is a data analysis and visualization software developed by Malvern Panalytical. It provides users with tools to import, analyze, and present data from various scientific instruments and experiments.
Automatically generated - may contain errors
Lab products found in correlation
Itc200 microcalorimeter, by Malvern Panalytical (7 mentions)
Microcal peaq itc, by Malvern Panalytical (2 mentions)
Microcal peaq itc analysis software, by Malvern Panalytical (2 mentions)
Vp isothermal titration calorimeter, by Malvern Panalytical (1 mentions)
Itc200 microcalorimeter, by GE Healthcare (1 mentions)
Vpviewer2000, by Malvern Panalytical (1 mentions)
Vp itc microcalorimeter, by Malvern Panalytical (1 mentions)
Itc200, by Malvern Panalytical (1 mentions)
12 protocols using origin software version 7
1
Quantifying FTSJ1-SAM Binding Interaction
2
Isothermal Titration Calorimetry of SLP65 and Vav Peptides
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ITC measurements were carried out on a VP-isothermal titration calorimeter, MicroCal, Inc (USA). The proteins were dialyzed for 24 hours against 20 mM Sodium Phosphate buffer, 100 mM sodium chloride, pH 6.0, followed by degassing. The SLP65 peptide solution was also prepared in the same buffer. All titration experiments were carried out at 25 °C using a syringe, constantly stirring the sample. In case of SLP65 peptide titration, stirring was carried out at 300 rpm, while the Vav SH3N titration was done at 200 rpm. For the binding isotherms, the protein solutions (20 μM of Grb2 SH3C for the peptide titration and 40 μM for the Vav titration) were titrated with 18–28 injections of the titrant (10ul of 600 μM Vav, and 10ul of 280 μM SLP65 peptide). Each injection was of 5 sec duration, followed by 4.5 min interval. The enthalpy change (ΔH) and binding constant (Ka) were obtained directly using Origin software, Version 7.0 (MicroCal, U.S.A). Other thermodynamic parameters were calculated using the formula ΔG° = RTlnKd.
3
Isothermal Titration Calorimetry of Lassa and Hantaan EN
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ITC measurements were performed at 25°C, using an ITC200 Micro-calorimeter (MicroCal, Inc). Experiments comprised 26 injections of 1.5 μL of 2.5–5.0 mM manganese or magnesium solutions into the sample cell containing 200 μL of 100–160 μM of Lassa EN (wild-type, E51A or D89A) or Hantaan EN (wild-type, E54G or D97A). All binding studies were performed in the lysis buffer. For data analysis the heat produced by the metal ion dilution in the buffer was subtracted from the heat obtained in the presence of the protein. Binding isotherms were fitted to a one-site binding or two-site sequential binding model using Origin Software version 7.0 (MicroCal, Inc). The initial data point was routinely deleted.
4
Calorimetric study of RNAP-ppGpp interaction
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An ITC-200
microcalorimeter
(GE Healthcare) was used for calorimetry studies. In brief, RNAP reconstituted
with and without ω/ω7/ω9 was
taken in the concentration range of 20–50 μM, and ppGpp
concentration was varied from 0.1 to 5 mM in all the experiments.
Both the ligand and protein were dissolved in a buffer consisting
of 20 mM Tris-HCl pH 7.9, 150 mM NaCl and 0.5 mM β-mercaptoethanol.
ORIGIN software Version 7.0 (Microcal, Malvern, Worcestershire, U.K.)
was used to calculate the binding affinity (Ka). At the time of plotting, the heat of dilution for ppGpp
was subtracted and the first data point was removed from the experimental
curve as suggested by the analysis software.
microcalorimeter
(GE Healthcare) was used for calorimetry studies. In brief, RNAP reconstituted
with and without ω/ω7/ω9 was
taken in the concentration range of 20–50 μM, and ppGpp
concentration was varied from 0.1 to 5 mM in all the experiments.
Both the ligand and protein were dissolved in a buffer consisting
of 20 mM Tris-HCl pH 7.9, 150 mM NaCl and 0.5 mM β-mercaptoethanol.
ORIGIN software Version 7.0 (Microcal, Malvern, Worcestershire, U.K.)
was used to calculate the binding affinity (Ka). At the time of plotting, the heat of dilution for ppGpp
was subtracted and the first data point was removed from the experimental
curve as suggested by the analysis software.
5
Determining hNSun6-SAM Binding Kinetics
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The equilibrium dissociation constants of the wild-type and mutant hNSun6 with SAM interactions were determined with an ITC200 Micro-calorimeter (MicroCal Inc.; Studio City, CA, USA). The binding enthalpies were measured at 25°C in 20 mM Tris–HCl, pH 7.0, 300 mM NaCl, 5 mM MgCl2 and 2 mM TCEP. The data were subsequently analyzed and fitted using a one set of sites model with Origin Software version 7.0 (MicroCal Inc.).
6
Quantifying ZBP-ZNP Interactions by ITC
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The affinity between a selected ZBP and ZNPs was determined by isothermal titration calorimetry (ITC). ITC was performed using a MicroCal VP-ITC Microcalorimeter with Origin software and VPViewer2000 (MicroCal Inc.). Titrations were performed by injecting 30 consecutive 10 μl aliquots of ZBP peptide (0.2 mM) into the ITC cell (volume = 1.4301 ml) containing nanoparticles (1 μM). Titration experiments were performed at 25 °C to determine the binding constant of ZBPs to the nanoparticle. The ITC data were corrected for the heat of dilution of the titrant by subtracting mixing enthalpies for 10 μl injections of ZBPs into nanoparticle-free solution. Binding stoichiometry, enthalpy, entropy, and equilibrium association constants were determined by fitting the corrected data to a bimolecular (One type of binding site) interaction model (Microcal Origin software version 7.0).
7
Characterization of THUMPD3 Binding to Sinefungin
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Isothermal titration calorimetry (ITC) measurements were performed at 25°C, using MicroCal PEAQ-ITC (Malvern Panalytical) or Isothermal titration microcalorimetry ITC-200 (Malvern Instruments). Experiments were performed by titration of 20 injections of 2 μl of sinefungin (SFG) (1 mM) into the Sample Cell containing around 50 μM purified THUMPD3 or THUMPD3–TRMT112 protein solution. The SFG and corresponding protein were hold in the same buffer C (50 mM Tris–HCl (pH 7.5), 300 mM NaCl). Titration of SFG to the same buffer was used as control to evaluated whether the titration system was normal or not. Binding isotherms were fitted by non-linear regression using MicroCal PEAQ-ITC analysis software or Origin Software version 7.0 (MicroCal). The ITC data were fitted to a one-site binding model using the two software as described as upon.
8
Isothermal Titration Calorimetry of SAH-Protein Interactions
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Isothermal titration calorimetry (ITC) measurements were performed using an ITC200 Micro-calorimeter (MicroCal Inc.; Studi City, CA, USA) at 25°C. Samples were buffered with 20 mM Tris–HCl (pH 7.5) containing 100 mM NaCl and 10 mM MgCl2. Aliquots of 1 mM SAH solutions (syringe) were injected step-wise into a 60 μM protein solution (cell). SAH titrated in an identical buffer was used as a control. Data were subsequently analyzed and fitted using a one set of sites model using Origin Software version 7.0 (MicroCal Inc.).
9
Binding Interactions of KANSL1 and MLL4 with WDR5
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ITC experiments were performed at 25°C using an ITC200 microcalorimeter (MicroCal). Experiments included 26 injections of 1.5 μL of 0.5 mM wild-type or 2 mM mutant peptide solution into the sample cell containing 50 μM WDR523–334 or WDS50–361 in 20 mM Tris (pH 7.0) and 200 mM NaCl. Peptides used were KANSL1 wild type (585-DGTCVAARTRPVLS-598-Y), KANSL1 R592A (585-DGTCVAAATRPVLSY-598), KANSL2 wild type (406-YEFSDDLDVVGDG-417), KANSL2 L411E,V413D (406-YEFSDDEDDVGDG-417), NSL2 wild type (155-YRDDDEIDVVSPH-166), and NSL2 I160E V162D (155-YRDDDEEDDVSPH-166). The initial data point was deleted from the data sets. Binding isotherms were fitted with a one-site binding model by nonlinear regression using Origin software version 7.0 (MicroCal).
To confirm that KANSL1 and MLL4 bind to the same binding site on WDR5, 0.56 and 0.35 mM MLL4 peptide (2504-LNPHGAARAEVYLRK-2518) was injected into the sample cell containing 50–60 μM WDR523–334 with or without 200 μM KANSL1 peptide, respectively. To test the mutually exclusive binding of KANSL1 and histone H3, 1.9 and 1.8 mM H3 peptide (1-ARTKQTARK-9-Y) was injected into the sample cell containing 120 μM WDR523–334 with or without 400 μM KANSL1 peptide, respectively.
To confirm that KANSL1 and MLL4 bind to the same binding site on WDR5, 0.56 and 0.35 mM MLL4 peptide (2504-LNPHGAARAEVYLRK-2518) was injected into the sample cell containing 50–60 μM WDR523–334 with or without 200 μM KANSL1 peptide, respectively. To test the mutually exclusive binding of KANSL1 and histone H3, 1.9 and 1.8 mM H3 peptide (1-ARTKQTARK-9-Y) was injected into the sample cell containing 120 μM WDR523–334 with or without 400 μM KANSL1 peptide, respectively.
10
Isothermal Titration Calorimetry of NSUN6
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Isothermal titration calorimetry (ITC) measurements were performed at 25 °C, using MicroCal PEAQ-ITC (Malvern Panalytical) or isothermal titration microcalorimetry ITC-200 (Malvern Instruments). Experiments were performed by titration of 20 injections of 2 μL 1 mM SAM into the sample cell containing around 50 μM purified NSUN6-D323N mutant protein solution. The SAM and corresponding protein were held in the same buffer (50 mM Tris-HCl (pH 7.0) and 100 mM NaCl). Titration of SAM to the same buffer was used as control. Binding isotherms were fitted by nonlinear regression using MicroCal PEAQ-ITC analysis software or Origin Software version 7.0 (MicroCal). The ITC data were fitted to a one-site binding model using the 2 software as described.
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