ࡱ> 9;8 @ 'bjbj .(؝؝ nnnnnnn* * * * F $f | | | | | | | $RC6Qn| | | | | 6nn| | | | | | n| n| | | | | nn| | Z * :| 0| 8 .| nnnnn| | | | | | | | | 66$f Isothermal titration calorimetry (ITC) 1. Introduction ITC is a technique which allows the progress of a binding reaction to be followed thermodynamically. When two substances bind, energy is either released or absorbed. ITC measures changes in binding enthalpy and entropy as this binding reaction occurs, allowing the calculation of binding constants and reaction stoichiometry, at any temperature between 2 and 80!. In general, ITC is used to measure the enthalpy change when a solution containing a macromolecule at known concentration. As the macromolecule within the analysis cell becomes saturated, the heat signal diminishes until only the enthalpy change associated with dilution is observed. Data analysis software is then used to obtain the desired thermodynamic data from the resulting curves. A useful introduction to ITC may be found on the MicroCal website: http://www.microcalorimetry.com 2. Safety There are no chemical hazards specific to use of the machine users should take all usual laboratory precautions appropriate to the chemicals being used, and according to COSHH. 3. Responsibility Initially, this instrument only be used following consultation with either Dr Teresa Pinheiro (B124,  HYPERLINK "mailto:T.Pinheiro@warwick.ac.uk" T.Pinheiro@warwick.ac.uk) and with direct supervision. Queries regarding maintenance and day-to-day upkeep should be directed to Ian Portman (Chief technician for the Structural Biology Group, Biological Science02476 522882). 4. Experimental Design For a thorough description of the technique and experiment design, users should refer to the operating manual. A rough guide is given here to allow users to asses whether a sample is suitable for analysis by ITC. The thermodynamic characterization of a reaction is dependent on the shape of a binding isotherm determined from the experimental data. The unit-less constant c (where c = KMtotn with K representing the binding constant, Mtot represents the concentration of macromolecule at the start of the experiment and n is the stoichiometry) determines the shape of this binding isotherm. This constant is a measure of how tight the binding is which is occurring in a reaction. Ideal values are 5 < c < 500, though other values are possible in the range 1 < c < 1000).  5. Sample guidelines In an ITC experiment aliquots of a titrant (protein, peptide or small molecule typically at e" 0.5 mM) are injected into the cell containing protein solution (typically 20 to 100M). Upon each titration the amount of heat released or absorbed is measured. In addition to the equilibrium dissociation constant (Kd), ITC titration allows to determine the number of binding sites, binding enthalpy and ent'()89:A    ĵ{j`Y`UC#hUh05B*CJ\aJphh0 hq5\h0h05\ h0h<>**䴳<B*CJaJphh{uh<h7^OJQJo(h7^ h0hU#h0hU5B*CJ\aJphh05B*CJ\aJph#h0h05B*CJ\aJphh0B*CJaJphhU5B*CJ\aJphh5vh05B*\ph'()9:  78O $dha$gdUgd< $dha$gd{u $dha$gd7^ dh7$8$H$gdU 7$8$H$gd0 $7$8$H$a$gd5v' $%RSTlm678AGNOP$ֲ󠑠~zrzrzc_[Lh0h0B*CJaJphh7^h}Ujh}Uh}UOJQJUh}Uh4H*h4hUhU5B*CJ\aJphhAI5B*CJ\aJph#hUhU5B*CJ\aJphhuh00JCJaJ,jhuh0B*CJUaJph jh0B*CJUaJphh0B*CJaJphhUB*CJaJphOP%&%%'''gd0 $dha$gd $dha$gd{u 7$8$H$gd0 $dha$gd4 $dha$gdUJL$8$9$:$$$~%%T'V'''''''{wh0hEB*CJaJphh0B*CJaJphh0h0B*CJaJphh{uhAI h{uH*Uh0h{uH* h0h{uh0hUB*CJaJphh{u6B*CJ]aJphh05B*CJ\aJph#hUh05B*CJ\aJphropy. ITC is a truly equilibrium solution method where Kd is measured for native proteins. No labeling or immobilization is required. Also, ITC is not limited by the ligand or protein size. It is relatively artefact-free, and is not affected by the optical properties of the samples. The only major disadvantage of ITC is that it requires relatively high concentrations of samples. No buffer or salt limitations. Except NO REDUCING AGENTS (DTT, etc) or unstable chemicals. Typically, common organic solvents (DMSO etc.) can be used. Importantly, the buffer composition should be EXACTLY THE SAME for the titrant and cell solutions. Good starting point is to use 50-100M protein (~2mls per titration) and 0.5-1 mM titrant (~300l per experiment). ,1h. 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