User`s manual

60 Chapter 6: Sample Experiments
Calorimetry Sciences Corp.
CSC 5300 N-ITC III 61
User’s Manual
Experiment Walk-Through
Introduction
This chapter contains descriptions of two well characterized titration experiments which
utilize materials readily obtainable and provide new users with the opportunity to develop
techniques and skills essential for the effective use of the N-ITC III. The rst experiment,
heat of protonation of Tris base may also be used as a chemical calibration to verify per-
formance of the calorimeter and its settings. The second experiment, binding of 2’-CMP
to RNase A, is included as an example of a typical well designed titration experiment.
Chemical Calibration
A chemical calibration tests all aspects of the instrument including the calibration con-
stant, the cell volume and the injection volume. There are several standard reactions
which are often used in calibrating isothermal titration calorimeters (see Briggner, L.-E.
and Wadsö, I. (1991) Test and Calibration Processes for Microcalorimeters, with spe-
cial reference to heat conduction instruments used with aqueous systems J. Biochem.
Biophys. Methods 22, 101-118.). Here we will describe one: protonation of Tris base
(Tris(Hydroxymethyl) Aminomethane). The Tris protonation experiment may be used to
determine or verify the calibration factor value setting used in the ITCRun software.
Heat of Protonation of Tris Base
Sample Preparation
Prepare a solution of Tris base by dissolving approximately 3 g in 100 mL of distilled
water. The solution will be approximately 250 mM, but the exact concentration is not
important so long as it is well in excess.
A 1.00 mM HCl solution is most readily prepared by pipetting 10 mL of standardized
0.1 N HCl into distilled water and diluting to 1 L in a volumetric ask. Alternatively,
a standard solution of HCl can be purchased commercially or standardized by acid-
base titration (see Skoog, D.A. and West, D.M. (1980) Analytical Chemistry (Saunders