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.
- 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.
- 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
- Best Practices for ITC to study binding interactions – Part 1
- 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
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