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:Malvern MicroCal ITC range

  • 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.

Table 1. Recommended instrument settings to run MicroCal ITCs.

More best practices will be in future blogs.

 

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