Easy Method Transfer from MicroCal VP-ITC to PEAQ-ITC

If you previously used a Microcal VP-ITC for binding studies, you can perform the same experiments on a MicroCal PEAQ-ITC. Use the experimental design wizard with the PEAQ-ITC software to predict ITC results with known parameters, and look at different concentrations and injection conditions.

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Introduction

Comparison of recommended ITC settings

Setting	VP-ITC	PEAQ-ITC
Temperature setting	2-80 °C. Most experiments performed between 10-37 °C	2-80 °C. Most experiments performed between 10-37 °C
Reference power	10-30 µcal/sec	3-10 µcal/sec
Stirring speed	Approx. 300 rpm	Recommended 750 rpm
Cell volume / volume needed to fill cell	1.4 ml/2 ml	200 µL/300 µL
Injection Syringe volume / volume needed to fill syringe	300 µL/500 µL	40 µL/75 µL
Range of injection volume	5-15 µL	1-3 µL
Typical injection conditions	1st injection 2 µL Injections 2-58: 5 µL OR injections 2-29: 10 µL OR injections 2-19: 15 µL	1st injection 0.4 µL Injections 2-39: 1 µL OR injections 2-19: 2 µL OR injections 2-13: 3 µL
Feedback	High	High
Filter period	2 sec	NA

Table 1: Comparison of recommended ITC settings

[Figure 2 TN240816-method-development-vp-itc-peaq-itc.jpg] Figure 2 TN240816-method-development-vp-itc-peaq-itc.jpg

Figure 1: PEAQ-ITC experiment simulator

Calculating sample concentration

Sample concentration depends on the binding affinity and the C parameter.

Assuming N = 1: C value = [Concentration in ITC cell]/KD

Best between 5 and 500
If needed, the C value can be down to 1 or up to 1000

For PEAQ-ITC, the minimum recommended concentration is 5-10 µM. For large heat change and/or tight binding, you can reduce the concentration to 1-5 µM. Weak binding and/or low heat changes need higher sample concentrations.

Sample concentration in ITC syringe

For 1:1 binding, use 10X the concentration in the ITC cell.

Since the PEAQ-ITC cell is smaller, a binding experiment in the PEAQ-ITC produces less heat change/injection and total heat change, compared to same titration performed with a VP-ITC. In many cases, the fit binding curve produces similar KD and ΔH results with both instruments (see figure 2 and table 2 below).

NOTE: It has been observed that if the ΔH is low, you may need higher sample concentration in the PEAQ-ITC, compared to the VP-ITC to achieve high-quality results.

[Figure 1 TN240816-method-development-vp-itc-peaq-itc.jpg] Figure 1 TN240816-method-development-vp-itc-peaq-itc.jpg

Figure 2: Left: PEAQ-ITC experiment with 0.1 mM EDTA in cell, 1 mM CaCl2 in syringe. Injection 1 0.4 µL. Injections 2-20 2 µL. N=1.01, KD 573 nM, ΔH -3.91 kcal/mole. Heat change for injection 2: -7.9 µcal. Total heat change: -78.2 µcal. Right: VP-ITC experiment with 0.1 mM EDTA in cell, 1 mM CaCl2 in syringe. Injection 1 2 µL. Injections 2-29 10 µL. N=1.03, KD 563 nM, ΔH -4.14 kcal/mole. Heat change for injection 2: -40.5 µcal. Total heat change: -562 µcal

Heat change guidelines for high-quality ITC data

Heat change	VP-ITC	PEAQ-ITC
Integrated Injection heat for 2nd injection (first full injection)	More than 10 µcal is ideal (minimum 5 µcal)	More than 2.5 µcal is ideal (minimum 1 µcal)
Total heat change for all injections	More than 80 µcal – below this value data can be noisier and result in less reliable fits	More than 16 µcal – below this value data can be noisier and result in less reliable fits

Table 2: Heat change guidelines for high-quality ITC data

ITC experiment design resources

For more guidelines on designing ITC experiments, please take a look at the resources below.