Proper sample preparation is essential for successful ITC testing. In particular, the minimal guidelines below must be strictly followed to insure an accurate estimate of stoichiometry (n), heat of binding (H), and binding constant (Kb) (or dissociation constant Kd = 1/Kb).
1.) The macromolecule solution (the sample to be placed in the reaction cell) must have a volume of at least 2.1 ml. The lowest concentration which can be studied is 3 M and this is adequate only for tight binding where Kd is smaller than 1 M. For weaker interactions, the macromolecule concentration should be 5 times Kd, or higher if possible. Preferably, the macromolecule solution should be dialyzed exhaustively against buffer for final equilibration.
2.) The ligand solution (the sample to be placed in the injection syringe) must have a volume of at least 0.7 ml. Its concentration should be at least 10 times higher than the concentration of macromolecule (if the macromolecule has multiple binding sites for ligand, then the ligand concentration must be increased accordingly). The buffer solution in which the ligand is dissolved should be exactly the same buffer against which the macromolecule has been equilibrated.
3.) After both solutions have been prepared, the pH of each should be checked carefully. If they are different by more that 0.05 pH units, then one of the solutions must be back-titrated so they are within the limit of 0.05 pH units. If any particles are visible in either solution, they should be filtered out.
4.) If possible, the concentrations of both solutions should be accurately determined after final preparation. Accurate determination of binding parameters is only possible if concentrations of binding components are known precisely.
5.) At least 20 ml of buffer must be sent along with the two samples, since this is used for rinsing the cell and for dilution if necessary.
6.) If possible, DTT should be avoided as a disulfide reagent and replaced by -mercaptoethanol or TCEP.
等温滴定微量热仪（ITC）基本介绍
等温滴定微量热仪（ITC）基本介绍
（美国 MicroCal ，美国微量热公司）
仪器设备名称：等温滴定微量热仪
制造国别：美国
制造厂商：美国微量热公司
规格型号：VP-ITC
品牌：MicroCal
总代理商： 华嘉（香港）有限公司
技术指标：
短期噪音水平：0.5纳卡/秒 (2 纳瓦)。
基线重复性：优于±20纳瓦/小时 。
工作温度： 2oC 到 80oC 。
最小响应时间： 17秒
可选择化学反应的响应时间： 多种选择（专利技术） 。
搅拌速度有多种选择。
控温方式：Peltier电子控温方式，快速达到控制温度，不需水浴。
样品池材质：铪合金 ，1.3 ml ， 固定式。
检测方式：功率反馈式,负反馈补偿，直接测定热量。
配置：
超灵敏量热仪主机， 工作软件， 进样、清洗装置， 基本附件。
仪器设备的先进性和实用性：
适用学科范围： 物理化学、生物化学、生物物理、化学生物学、生命科学、药物学、土壤学、胶体化学 等。
该仪器的特点和优势 ：
1.该型号仪器的检测原理为功率反馈式, 功率负反馈补偿，直接测定热量， 测得的结果直接， 准确 。
2.仪器的灵敏度最高，短期噪音水平0.5纳卡/秒 (2 纳瓦)， 样品量可以低至微克级 。
3.该仪器可选择化学反应的响应时间， 从而能提供研究多样性反应过程的可能性。
4.该型号仪器使用时无其它消耗品需求， 不需固定化、不需修饰， 对包括混悬液、带颜色的样品、一定黏度的样品体系等都可适用。
5.该型号仪器的控温方式为Peltier电子控温方式，可快速达到控制温度，不需水浴， 控温精度高。
6.该型号仪器的软件功能全面，专业的控制和数据处理软件（VPViewer ， Origin 7.0 ）包含目前所发现的全部反应模型和数据解析模型。
7.全球引用该仪器产生的数据而发表的文献， 占同类文献的份额为95%以上。
该仪器设备对环境的要求：洁净， 避免强光、电场、磁场、气流波动、震动。
