ITC kinetic experiments (enzyme activity)

Work of the Gygli group...

Investigation 1: An experimental workflow to provide detailed information of the molecular mechanisms of enzymes is described. This workflow will help in the application of enzymes in technical processes by providing crucial parameters needed to plan, model and implement biocatalytic processes more efficiently. These parameters are homogeneity of the enzyme sample (HES), kinetic and thermodynamic parameters of enzyme kinetics and binding of reactants to enzymes. The techniques used to measure ...

An experimental workflow to provide detailed information of the molecular mechanisms of enzymes is described. This workflow will help in the application of enzymes in technical processes by providing crucial parameters needed to plan, model and implement biocatalytic processes more efficiently. These parameters are homogeneity of the enzyme sample (HES), kinetic and thermodynamic parameters of enzyme kinetics and binding of reactants to enzymes. The techniques used to measure these properties are ...

ITC multiple injection (MIM) experiments to determine the kinetic parameters of NDK (nitrononane-2,8-dione) and NADPH with Gre2p in 100 mM KPi buffer pH 7.5 Data from ITC recurrent single injection (rSIM) experiments were used to achieve proper fitting of kcat values.

ITC multiple injection (MIM) experiments to determine the kinetic parameters of NDK (nitrononane-2,8-dione) and NADPH with Gre2p in 1x PBS buffer pH 7.5 Data from ITC recurrent single injection (rSIM) experiments were used to achieve proper fitting of kcat values.

ITC multiple injection (MIM) experiments to determine the kinetic parameters of NDK (nitrononane-2,8-dione) and NADPH with Gre2p in 100 mM HEPES buffer pH 7.5 Data from ITC recurrent single injection (rSIM) experiments were used to achieve proper fitting of kcat values.

ITC recurrent single injection (rSIM) to determine the kinetic parameters of NDK (nitrononane-2,8-dione) and NADPH with Gre2p in 100 mM KPi buffer, 1x PBS buffer and 100 mM HEPES buffer, all with pH 7.5

Python workflow for the analysis of ITC-BIND, ITC-MIM and ITC-(r)SIM experiments. Organized in a *.zip folder. Requires the following directory structure:

./ITC_analysis.py ./input/BINDING/.apj ./input/BINDING/.csv ./input/KINETICS/.apj ./input/KINETICS/.csv ./scripts/binding_neu.py ./scripts/kinetics_neu.py

And can be executed by running python ITC_analysis.py in the directory. Filenames for the input *.apj and *.csv files are defined in ITC_analysis.py. The output directory is written by ...

ITC multiple injection (MIM) experiments to determine the kinetic parameters of NDK (nitrononane-2,8-dione) and NADPH with Gre2p in 0.1%Tween-20-100 mM KPi buffer pH 7.5 Data from ITC recurrent single injection (rSIM) experiments were used to achieve proper fitting of kcat values.

ITC recurrent single injection experiment for the reaction of NADPH and NDK with Gre2p in KPi, PBS and HEPES buffer (100, 1x, 100 mM, respectively, all at 25°C and pH 7.5). In this apj file, the data for the analysis of the first injection peak is stored. *.apj file

ITC recurrent single injection experiment for the reaction of NADPH and NDK with Gre2p in KPi, PBS and HEPES buffer (100, 1x, 100 mM, respectively, all at 25°C and pH 7.5). In this apj file, the data for the analysis of the second injection peak is stored. *.apj file

ITC recurrent single injection experiment for the reaction of NADPH and NDK with Gre2p in KPi, PBS and HEPES buffer (100, 1x, 100 mM, respectively, all at 25°C and pH 7.5). In this csv file, the data for the analysis of the both injection peaks is stored. *.csv file

ITC multiple injection experiment for the reaction of NADPH and NDK with Gre2p in 0.1%Tween-20-KPI buffer (100 mM, pH 7.5) at 25 °C. *.apj file

ITC multiple injection experiment for the reaction of NADPH and NDK with Gre2p in 0.1%Tween-20-KPI buffer (100 mM, pH 7.5) at 25 °C. *.csv file

ITC multiple injection experiment for the reaction of NADPH and NDK with Gre2p in HEPES buffer (100 mM, pH 7.5) at 25 °C. *.apj file

ITC multiple injection experiment for the reaction of NADPH and NDK with Gre2p in HEPES buffer (100 mM, pH 7.5) at 25 °C. *.csv file

ITC multiple injection experiment for the reaction of NADPH and NDK with Gre2p in KPI buffer (100 mM, pH 7.5) at 25 °C. *.apj file

ITC multiple injection experiment for the reaction of NADPH and NDK with Gre2p in KPI buffer (100 mM, pH 7.5) at 25 °C. *.csv file

ITC multiple injection experiment for the reaction of NADPH and NDK with Gre2p in PBS buffer (1x, pH 7.5) at 25 °C. *.apj file

ITC multiple injection experiment for the reaction of NADPH and NDK with Gre2p in PBS buffer (1x, pH 7.5) at 25 °C. *.csv file

SOP describing how to perform an ITC multiple injections experiment with a MicroCal PEAQ ITC by Malvern.

SOP describing how to perform an ITC (recurrent) single injections experiment with a MicroCal PEAQ ITC by Malvern.

Python workflow for the analysis of ITC-BIND, ITC-MIM and ITC-(r)SIM experiments. Organized in a *.zip folder. Requires the following directory structure:

./ITC_analysis.py ./input/BINDING/.apj ./input/BINDING/.csv ./input/KINETICS/.apj ./input/KINETICS/.csv ./scripts/binding_neu.py ./scripts/kinetics_neu.py

And can be executed by running python ITC_analysis.py in the directory. Filenames for the input *.apj and *.csv files are defined in ITC_analysis.py. The output directory is written by ...

SOP describing the steps for use of the ITC device (MicroCal PEAQ ITC by Malvern).