Best Practices for Isothermal Titration Calorimetry to study binding interactions – Part 2

Here is the second best practices blog for performing traditional binding experiments with the MicroCal PEAQ-ITC, VP-ITC and ITC200 systems. 

Here is the first one: Best Practices for ITC to study binding interactions – Part 1

Preparing the ITC:

• Perform a visual inspection of the ITC system, and check for loose cable connections, damaged injection syringe, leaky tubing connections, discoloration around the cell, etc.
• Make sure water to the ITC reference cell, and replace water regularly during the use of the ITC (once a week is recommended)
• Make sure the ITC and controller are powered on, the control software is opened, the instrument is on-line, and the instrument thermostat is set to the experimental temperature.
  • MicroCal ITCs can be left on indefinitely and thermostatted at 25 °C
• Clean the ITC cell and syringe, using recommended cleaning protocol and reagents
  • Recommend rinsing the ITC cell with 20% Contrad 70/14% Decon 90, followed by water, after each ITC experiment.
  • Perform regular “soaks” with 20% Contrad 70/14% Decon 90 in the ITC cell, heated to 60 °C, for at least 30 minutes. Cool the cell and rinse with water. If you are working with a sticky protein or other biomolecule, you may need to do more frequent detergent soaks.
  • Make sure the ITC syringe is rinsed with methanol and dried before adding the ligand.
  • You can also rinse the ITC syringe with several volumes of ligand solution.
• Fill the ITC cell and syringe, using the recommended filling and loading protocols. It is not necessary to degas the samples when using the PEAQ-ITC and the ITC200
  • Rinse the ITC cell with sample buffer, before filling the ITC cell with sample.

ITC settings:

• When using ITC data for screening and comparison of affinity and thermodynamic parameters, be sure all the experimental design settings are the same, including temperature, feedback mode, and injection conditions.
• See Table 1 for the recommended settings.

For automated MicroCal PEAQ-ITC and Auto iTC200 systems:

• Be sure the samples are stored at an appropriate temperature prior to ITC analysis. For proteins and other biomolecules, set the plate storage tray temperature to 5 to 10 °C. If you are studying a thermostable material, you can set the tray to 25 °C.
• Program cell and syringe cleanings using 20% Contrad 70 or 14% Decon 90, using appropriate automation routines and during regular maintenance.

More best practices will be in future blogs.

Relevant content:

• Isothermal Titration Calorimetry: Theory and Practice
• Practical tips for MicroCal PEAQ-ITC experiments
• Addressing complexity of binding interactions with ITC – get the most out of your ITC data
• Biomolecules: sample and data quality in interaction analysis – Two sides of the same coin
• Don’t throw away your “bad” N-value ITC data
• Gold standard for binding affinity

Previous posts:

• Best Practices for ITC to study binding interactions – Part 1
• Best Practices for ITC to study binding interactions – Part 3
• Best Practices for Isothermal Titration Calorimetry to study binding interactions’ part 4
• Isothermal Titration Calorimetry – A hot characterization tool for biomolecular interactions
• Isothermal Titration Calorimetry: An essential tool for binding affinity measurements in life sciences research
• What’s new in ITC? June 2018 edition