MicroCal Auto-iTC200 system

This Technical Note offers a brief technical overview of MicroCal Auto-iTC200 system and applications.

The Malvern MicroCal Auto-iTC200 isothermal titration calorimetry (ITC) instrument allows direct and label- free measurement of binding affinity, and thermodynamic parameters. Heat released or absorbed during biochemical binding events is measured directly, giving information about binding affinity (KD), stoichiometry (n), enthalpy (ΔH), and entropy (ΔS) in a single experiment. This data reveal the forces that drive complex formation, providing deeper insights into structure-function relationships and the mechanisms of binding. In drug discovery, the information can be used together with structural data to guide drug design and to resolve the mechanism of drug action.

MicroCal Auto-iTC200 combines the exceptional performance of MicroCal iTC200 with full automation for unattended operation addressing the needs of busy research and drug discovery laboratories.

Key benefits

  • All binding parameters (affinity, stoichiometry, enthalpy and entropy) in a single experiment.

  • Quick to first result with minimal assay development, no labelling, no immobilization and no molecular weight limitations.

  • Sensitivity to investigate any biomolecular interaction using as little as 10 μg of protein.

  • Exceptional data quality for sub-millimolar to picomolar affinity constants. Directly measures millimolar to nanomolar binding constants (10-2 to 10-9 M). Measures nanomolar to picomolar disassociation constants using competitive binding techniques (10-9 to 10-12 M).

  • Fully automated system with walk-away operation for enhanced productivity.

  • User-friendly experimental design wizards and automated data analysis for fast and reliable analysis.

Fig 1. MicroCal Auto-iTC200

With MicroCal Auto-iTC200, sample cell filling, injection, and cell cleaning functions are fully automated. The microcalorimeter used in MicroCal Auto-iTC200 is the same as that used in MicroCal iTC200 with the same fast equilibration and response times. The sample and reference cells in MicroCal iTC200 are made from Hastelloy™ alloy, a nonreactive material chosen because of its excellent chemical resistance and applicability with biological samples. Three different user-selectable titration response times (feedback modes) provide application versatility, and variable mixing speed facilitates the use of a variety of sample types. A temperature-controlled sample tray in MicroCal Auto-iTC200 holds up to four 96-well plates and can be maintained to 4°C.

All binding parameters in a single experiment with minimal assay development

ITC measures binding affinity (KD), stoichiometry (n), enthalpy (ΔH), and entropy (ΔS) of the interaction of the binding partners in their native state without requiring modification of the components with fluorescent tags or requiring immobilization. Other than preparing samples and reagents to appropriate concentrations, there is no assay development needed and the time to first result is fast.

Simultaneous determination of thermodynamic parameters for reaction mechanism elucidation

To study the interaction of carbonic anhydrase with five known inhibitors with the purpose of elucidating the reaction mechanism, a series of 20 runs was performed (Table 1). The workflow, including sample introduction, titration, and cleaning was completely automated. Each inhibitor was run four times and all 20 runs were completed in under 24 hours (Fig 2). Both CBS and furosemide have similar binding affinities, yet different enthalpies, suggesting that the binding mechanisms are different.

Table 1. Thermodynamic parameters determined for the interaction of five inhibitors with bovine carbonic anhydrase II (BCA). The values are the average of four separate runs with errors shown
Ligand/ Concentration BCA (µM) n KD(µM) ∆G (kcal mol-1) ∆H (kcal mol-1) -T∆S (kcal mol-1)
0.126 mM
10 0.98 ±0.02 0.06 -9.87 -11.15 ±0.46 1.28
0.414 mM
30 1.00 ±0.04 0.96 -8.21 -10.19 ±0.12 1.98
0.426 mM
30 0.98 ±0.08 0.92 -8.23 -7.06 ±0.20 -1.17
0.441 mM
30 0.99 ±0.05 4 -7.35 -7.93 ±0.39 0.58
0.525 mM
30 1.03 ±0.02 0.35 -8.8 -2.03 ±0.07 -6.77

The binding signatures for these interactions are shown in Figure 3. From the enthalpy and unfavorable entropy factor, CBS binding is driven primarily by hydrogen and van der Waals bonds with some conformational changes reducing the affinity. Furosemide has a more balanced binding based on hydrogen and van der Waals bonds as well as hydrophobic effects. This illustrates that even when the inhibitors have almost identical binding affinities, important differences in binding mechanisms can be easily determined.

Fig 2. Overlay plots from four repeated titrations of bovine carbonic anhydrase II, with CBS (left) and furosemide (right).
Fig 3. Binding signatures for five inhibitors of bovine carbonic anhydrase II.

Outstanding sensitivity and data quality gives confidence in results

The sensitivity of MicroCal Auto-iTC200 provides excellent signal-to-noise to generate high quality data even at the low protein concentrations required for high affinity interactions or so called “tight binders.”

When studying tight binders, the experimental challenge is using the minimum protein concentration that will produce a heat change that can be measure with good signal-to-noise. A 1 µcal injection with excellent signal-to-noise is shown in Figure 4. There is a relatively low concentration of protein, 3 µM carbonic anhydrase in the sample cell where the typical recommended protein concentration is 10 µM. Even at a protein concentration of 3 µM, a strong signal is generated with MicroCal Auto-iTC200.

Fig 4. Carbonic anhydrase (3 µM) titrated with CBS (4-carboxybenzene sulfonamide)

Precise automation generate reproducible, high quality data

Excellent sensitivity combined with the precise automation of MicroCal Auto-iTC200 yield exceptional reproducibility and high quality data. This is demonstrated by the small relative standard deviations of the values for stoichiometry and enthalpy for the interaction of carbonic anhydrase with five inhibitors (Table 1) and by the overlays of replicate injections of the furosemide and CBS titrations presented in Figure 2.

Experiment across a wide range of affinities

The excellent sensitivity and precise operation of MicroCal Auto-iTC200 permits experimentation over a wide range of affinities from weak to tight binders. For example, the binding thermodynamics for 11 inhibitors to human CA2 was studied using MicroCal Auto-iTC200. The raw data as well as integrated heats from the interaction of human CA2 and 4-carboxybenzenesulfonamide are shown in Figure 5. The calculated values for affinity and enthalpy for the 11 inhibitors are in Table 2. The affinities for the 11 substances binding to human CA2 span a 5000-fold difference in affinities.

Fig 5. Raw data (top) and integrated heats (bottom) from the titration of human CA2 (29 µM) with 4-carboxybenzenesulfonamide (297 µM). The solid line through the integrated heats represents the best fit binding isotherm to a one-to-one binding model using the Origin™ software provided with the instrument.
Table 2. Affinities and enthalpies for 11 substances binding to human CA2
No. Compound KD
(kcal mol-1)
1 Ethoxzolamide 0.00083 - 14.4 10 107
2 Acetozolamide 0.013 - 14.7 10 97
3 Methazolamide 0.023 - 13.0 10 115
4 4-nitrobenzenesulfonamide 0.093 - 13.8 10 100
5 p-toluenesulfonamide 0.58 - 10.0 29 282
6 Azosulfamide 0.49 - 17.6 29 312
7 Benzenesulfonamide 1.4 - 10.8 29 309
8 4-carboxybenzenesulfonamide 0.84 - 15.0 29 297
9 2-aminobenzenesulfonamide 1.9 - 12.0 29 273
10 Furosemide 1.3 - 4.2 30 255
11 Sulfanilamide 4.0 - 10.1 29 405

Fully automated with walk-away operation drives productivity

MicroCal Auto-iTC200 is composed of the microcalorimeter, a fluidic system, an autosampler, and temperature-controlled tray for storing samples. The fluidic system includes a series of robotic arms and liquid transfer devices for moving sample and reagents to and from the calorimeter cell for fully automated sample cell loading and cell cleaning. The sample tray that holds up to four 96-well plates can be maintained between 4°C and ambient temperature. Running an experiment is as easy as setting up the experimental conditions and sample list in the software, filling the plates with sample, loading the plates in the tray, pushing “start” and walking away to engage in other more laborious activities.

Fig 6. This illustration shows the Plate Setup tab when loading both the cell and the pipette from a 96-well plate with the option of saving the sample enabled for sample groups 1, 2, 3 and 10.

Optimized methods and automated data analysis enhances data quality

MicroCal Auto-iTC200 instrument control and data analysis software enhances data quality and streamlines data analysis. The control software incorporates optimized scripts for sample equilibration to minimize the occurrence of data artifacts resulting from sample outgassing and for fluidic component and cell cleaning preventing carryover. The data analysis software has a robust baseline fitting algorithm eliminating the need for tedious manual manipulation post-run. Experimental controls are automatically subtracted saving time.


The ability to measure KD, n and ΔH values using a simple assay without the need for labels or immobilization and the ability to automate for walk-away operation has led to the broad uptake of MicroCal Auto-iTC-200 among drug discovery and development groups performing hit validation, mechanism of action studies, and lead optimization. It is also used to measure protein activity as a batch-to-batch QC test and as a tool for the development of higher throughput assays.

The types of interactions that can be studied are not limited to proteins. There are numerous references in the literature where ITC has been used to understand how nucleic acids and lipids, as well as proteins, function in biological systems.

Validate fragment hits from mM to µM affinities in a day with minimal assay development

It is important to identify false positives from a primary screen at an early stage so that further time is not spent investigating these compounds. MicroCal Auto-iTC200 data was used to validate hits from a thermal shift-based primary screen and to accurately rank the affinities so that only the tightest binders were put forward for co-crystallization trials and the structure based medicinal chemistry program.

Of the 47 fragments selected from the thermal screen, 33 provided good binding data that could be used to quantitate the affinity and 14 showed either no heat of binding or unusual isotherms (Fig 7). The 33 promising fragments were ranked in order of affinity and the 20 strongest binders were rescreened to assess the robustness of the affinity determination.

Fig 7. Examples of fragments analyzed with MicroCal Auto-iTC200. The left and the right graphs show raw data (upper panels) and binding isotherms (lower panels) of a fragment with an affinity of approximately (A) 0.9 mM and (B) 4 µM. Of these compounds the stronger binder was selected for further study.

Accurate binding energetics to support structure-based lead optimization

Drugs should bind to targets with high affinity and selectivity. Traditionally, lead optimization has been driven by studies of the affinity component. Thermodynamic properties (ΔH, ΔS) are also fundamental to binding and can provide deeper insights into the interactions. The sensitivity and walk-away automation of MicroCal Auto-iTC200 allow efficient determination of accurate KD values and enthalpy and entropy to support structure-based lead optimization.

Fig 8. Modeling suggested that addition of an ether group to lead compound would allow the compound to extend into a pocket in the target protein increasing affinity. The ITC data indicate the addition has more favorable enthalpy, less favorable, and 7-times greater affinity.


The data in Figure 8 is abstracted from Sarver, Current Trends in Microcalorimetry.

Technical specifications

Sample volume 370 μL
Equilibration time from 25°C to 5°C < 6 min
Response time 10 s*
Sample capacity 384 (four 96-well plates)
Throughput 22 per 24 h
42 per 24 h (SIM)
Sample tray temp. range 4°C ± 2°C to ambient
Injection volume precision < 1% at 2 μL
Injection syringe volume 40 μL
Cell material Hastelloy
Cell configuration Coin-shaped
Cell volume 200 μL
Noise 0.2 ncal/s
Temperature range 2°C to 80°C
Temperature stability at 25°C ± 0.00015°C
Multiple feedback modes Yes (passive, high gain, low gain)
Dimensions, calorimeter
(W × H × D)
62.5 × 76.8 × 57.2 cm
Weight 90.7 kg

* High feedback

Using typical run parameters (cell cleaning with detergent, 12 × 3 µL injections)

Cell temperature = 25°C, no feedback, 5 s filter period, stir speed = 1000 rpm


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