这项工作是支持由NSF资助MCB - 0817827。

等温滴定量热（ITC）是一个完善的技术，可以决定在一个实验中的所有一项具有约束力的相互作用的热力学参数（亲和力，焓和化学计量学 ），1国贸中心工程由滴定成为第二个反应物，在等温条件下的反应物。测得的信号是两反应物的相互作用（绑定）后释放或吸收的热量。一系列的注射执行和限制反应物趋于饱和，热信号将趋近于零。等温线的拟合给出的热力学参数。一些评语是可用的仪器仪表以及数据收集和分析的数学描述。2,3虽然其他热量计（最显着的贸易中心与小卷 200），在这里，我们描述了一个一般的VP - ITC协议制造MICROCAL（现为GE医疗集团的一部分）。 1。准备样品<ol><li>为了准备和缓冲高分子配体，一些潜在的问题需要加以解决。准确的适合需要适当浓度的高分子，通常在国际贸易中心的样品池，并配体的物种，在注射器的物种。由于某些蛋白质聚集在注射器中的物种所需要的高浓度，最常见的蛋白质是装入样品池。从“C”，产品的预测系统的亲和力，它可以采用正交的方法，使用国际贸易中心前估计，总高分子浓度，确定最佳的高分子浓度， 其中C = K A * [M]。 C范围内的最优值从10-1000，虽然它是弱结合系统的C -值低于下限具体的实验条件下得到准确的数据 。因此，高分子浓度必须确定此范围记C值（即10 6米-1的K，应采用高分子浓度10〜1000微米）。之前系统结合的亲和力知识可以帮助减少贸易中心的使用，通过更好的ITC实验设计蛋白质。配体的浓度应足够大（比K ð集中最弱的配体结合位点的7-25倍以上），饱和度，使发生在最初的三分之一到一半滴定。还需要准确的数据拟合信号的饱和度。对于具有较高的结合亲和力系统中，配体浓度较低的，应使用以避免饱和为时尚早滴定，这将给不准确的适合。一旦已减去滴定稀释控制热量（即到缓冲区配体滴定），在饱和焓应该趋近于零。 </li><li>由于小分子杂质，可能会引起在国贸中心测量的artifactual信号，它是最好的，如果可能的话，大分子与配体尽数对缓冲区透析。另外，柱层析，脱盐离心柱，或缓冲液交换离心式过滤器（例如，Centricons）可以用来改变大分子的缓冲区。如果配体是一种小分子，它可以使用大分子后已与合适的截止时间为小分子（即100-​​500大一个光谱/ POR浮法一个仪透析对透析膜透析或透析缓冲准备）。缓冲液成分之间的配体和高分子解决方案的差异可能会导致信号稀释样品中杂质的热工件。经过准备，确保检查的缓冲区，高分子和配体匹配的pH值在焓文物（± 0.05 pH单位）可以引起缓冲质子效果。5确保足够的准备，每一个物种。一式三份实验和稀释控制，所需材料的数量将取决于所使用的国际贸易中心。但是，对于大多数的国贸中心仪器约2毫升的量样品细胞，至少高分子溶液6-7毫升将需要，并可以方便地在15毫升隼管准备。 1-2溶液300μL注射器，应足够，并可以在猎鹰管或离心管准备。 </li><li>灰尘和其他微粒，可引起工件在国贸中心温度记录的基准。当务之急是，他们被删除之前运行实验。编制样本股的解决方案后，样品应在离心管离心五分钟到溶液中的沉淀颗粒在8000到14000 RPM。去除上清，小心，不要去打扰颗粒，并将其放置在一个新的猎鹰/离心管。 如果需要保持降低半胱氨酸还原剂是，使用低浓度的β-马尔卡和新鲜的股票ptoethanol，TCEP（三（2 - 羧基）膦）或二硫苏糖醇，以尽量减少由于还原剂氧化的工件。此外，在缓冲区的存在有机溶剂（常见的一些可能需要甲醇或DMSO以可溶性的小分子配体）可能会导致信号的文物。如果需要配体的有机溶剂，然后高分子解决方案还必须包含相同的浓度，以避免有机溶剂的稀释热所产生的任何信号。在缓冲区之间的配体和高分子因夹带剂成分的细微差别，盐或pH值是可能的样品制备过程中。最好的方法是检查进样缓冲或大分子的样品缓冲液的配体的稀释，以确保从缓冲区中的内容的差异所产生的热量信号不会导致数据单纯从文物产生。 </li><li>检查高分子配体和谨慎地使用适合您的系统技术（如吸光度测量，高效液相色谱法，比色法，BCA法测定蛋白质等），记录它们的确切浓度的浓度。在实际浓度和使用，以适应等温线的浓度的差异会导致在化学计量学，焓和有约束力的亲和力，从实验确定的错误。德加样品，以避免由于气泡或溶解的气体在滴定释放的信号文物，特别是在较高温度下，是很常见的。 </li></ol> 2。设立实验<ol><li>确保样品池和注射器是根据制造商的协议清洗的装货前对大分子与配体。 1.8毫升蒸馏水使用​​汉密尔顿注射器（使用保健为每桶容易折断与汉密尔顿注射器或针头弯曲）的水冲洗样品池两次或三次。下一步冲洗样品池1.8缓冲液数次。高分子溶液1.8毫升，装入样品池，小心，以避免泡沫的形成。 </li><li>用蒸馏水填充引用单元格。对于大多数缓冲区，蒸馏水是正常使用，作为对照品溶液。然而，对于具有特别高离子强度或渗透压的缓冲区，这是更好地利用缓冲区作为参考。 </li><li>取出的样品使用汉密尔顿注射器参考和细胞的气泡。向上和向下的细胞敲任何气泡，可在单元格的底部和连接井细胞顶部的两侧，轻轻移动注射器针头。删除任何过剩体积的样品和参比细胞。 </li><li>将一个塑料注射器的使用管注射器注射的填充端口。用蒸馏水冲洗注射器。按照缓冲区冲洗。确保注射器是完全按图纸通过系统内的空气疏散。放入配体的解决方案，注射器的针头和借鉴注射器配体的解决方案，直到整个注射器。继续绘制到的端口和连接管一点点多余的量（约50μL或更多）。立即关闭注射器填充端口和分离管和塑料注射器。清除和填充注射器两次从注射器中删除任何气泡。从配体解决方案中删除的注射器和一个kimwipe，以消除任何滴擦拭一边，小心不要触摸注射器尖端的kimwipe因为这可能会删除从注射器的体积。此外，要小心不要敲或jar注射器，这也可以造成损失量从注射器尖端。注射器放入样品池。 </li><li>成立国际贸易中心的运行参数。对于具有较强的热信号约束力的系统，大量的低容量注射剂将给予更多的数据点（如75注射3μL）装修。系统有弱热信号，少数的大容量注射剂是可取的（如33 8μL注射）。最常见的，具有较高的注射量较少注射使用。这可能需要几个滴定，以优化的条件，是最适合您的系统。重要的是要注意的，具有约束力的焓可以放热或吸热，取决于正在研究的系统。不幸的是，有些系统具有低热量信号，使反应热难以确定。这种系统的问题可能会被克服增加大分子的浓度，改变温度的实验（取决于绑定系统的热容量），和/或改变pH值或离子强度的缓冲。 </li><li>同时考虑每次注射之间的时间间隔。当务之急是，每个配体注射后，系统给定的时间来平衡和热信号返回未来注入发生前的基线。对于大多数系统，三到五英里nutes应该足够。之间的注射时间应增加系统的平衡，不会发生在五分钟内。 </li><li>因为会有一些混合，一旦之间的高分子配体的解决方案和注射器插入进样细胞，第一次注射会给假的结果。最好使用小卷的第一个或两个注射剂（使他们可以在以后被丢弃）（如2μL）和所需的值保持在随后的注射。 </li><li>选择实验的温度（以25 ° C是最常见的，虽然在2和80之间° C的温度可以使用）。这是最好的选择匹配高分子配体系统上执行的其他实验（绑定，动力学等）的温度。国际贸易委员会可设立在不同的温度，从实验的温度平衡。如果试验是在温度超过10℃远离室温，那么最好设置为平衡温度在5 ° C的实验温度的仪器。这将减少时间仪器将达到实验的温度。 注射器的搅拌速度也需要考虑。搅拌足够的滴定过程中的配体和高分子混合是必要的，但一些蛋白质是由不稳定的快速搅拌。对于这些情况，搅拌速度应设置在一个相对较低的利率。一旦所有的实验参数已经成立，那么就可以开始实验。 </li><li>一旦实验完成后，ITC可根据制造商的协议清洗。如果所需的额外的高分子配体的复杂混合物的实验样品中的细胞在实验结束的解决方案，可以保持。 </li><li>重复滴定至少有一个或两个以上的时间得到重复性的数据。运行控制配体是一个缓冲区，以确定为配体的稀释热滴定样品中的细胞。对于一些有约束力的合作更多的信息，对绑定过程的系统中，可以得到注入高分子配体。不同注射方向可以提供更多的信息，这可能有助于全球拟合。 </li></ol> 3。分析数据<ol><li>数据拟合，可以很容易地使用宏在任何数据拟合程序（通常是随着仪器的制造商提供）。装入第一个数据文件。检查原料温度记录信号中的气泡或其他文物的任何迹象。如果有任何构件（如当时没有在这个时间点注射在基线或峰峰值），然后记下这些数据点，因为他们应该被删除。接下来，负载配体稀释控制数据，并减去配体的结合等温线稀释数据。此时，删除所有虚假的数据点，包括第一个或两个数据点发生稀释文物的滴定（见步骤7）。 </li><li>选择的数据拟合模型（结合位点，两个/多个结合位点，合作约束力等）被用来拟合数据。的数据可以配合的拟合参数，化学计量学（N），焓（ΔH）和亲和力（K 一 ）初始猜测。如果结合的亲和力，或其他参数的先验知识，是从已知的正交实验，然后可以输入这些值。这有助于避免的契合，成为被困在装修局部极小。一旦数据已符合第一滴定，重复步骤15和16等温线的其余部分。另外，数据可以适合全球使用SEDPHAT，如方案6 SEDPHAT可能是二进制复杂的数据集，结合三元复杂的数据集（即分子的A + B）的全球装修，特别是在有帮助（分子通过A + B + C）。例如，在C到一个AB的复杂的分子具有约束力，它并不总是很清楚是否有可能是由于C绑定的A或B的一个（如果A是不完全饱和）的任何信号。 </li><li>为了确保国际贸易委员会的数据是不artifactual，应该从ITC得到的结合亲和力和化学计量比正交法7,8此外，ITC焓值可以比较从范特霍夫积焓。9它也可以证明是有益的的，使用不同浓度的配体或高分子的热信号的绝对值随着高分子浓度的增加应增加。文物最有可能出现在细胞和注射器的缓冲区，如果不匹配。另一个构件的可能性来自不纯的样品。 </li><li>我们建议用户用简单的二进制复杂滴定开始，其中有以前的信息对K d和stoich提供iometry。然后，如果额外的配体结合，用户可以尝试更复杂的三元复合滴定法，不同的配体浓度，也许交换注射器和电池组件。为三元复合物的形成路径的焓的比较应添加剂作为焓是状态函数。，10，SEDPHAT 6可能会非常有用。 </li></ol> 4。代表性的成果： <img alt="图1" src="/files/ftp_upload/2796/2796fig1.jpg" /> 一个有代表性的例子为辅酶NADPH结合到 E乖巧的滴定图1 。 大肠杆菌染色体二氢叶酸还原酶（ecDHFR）。面板（*）显示的原料温度记录;，从集成热像适合的结合等温线（二）使用Origin软件在一个网站的模式;及（C）等温线的拟合使用一个具有约束力的网站模式，从SEDPHAT沿合适的残差。从原点软件，使用的一个网站绑定模型，N = 1.09 ± 0.02，K D = 0.194 ± 0.001μM，ΔH= -22.7 ± 0.4千卡/摩尔，TΔS= -13.1 ± 0.4千卡/摩尔，和ΔG= - 9.16 ± 0.01千卡/摩尔。适合数据使用Sedphat负担0.94 Ñ ± 0.01，K ð的0.195 ± 0.013ΔM，ΔH为-22.5 ± 0.2千卡/摩尔，-13.39TΔS千卡/ mol和-9.15千卡/摩尔的ΔG。

<ol>
	<li>Wiseman, T., Williston, S., Brandts, J. F., Lin, L. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+measurement+of+binding+constants+and+heats+of+binding+using+a+new+titration+calorimeter.">Rapid measurement of binding constants and heats of binding using a new titration calorimeter.</a> Anal. Biochem. 179, 131-137 (1989).</li><li>Jelesarov, I., Bosshard, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isothermal+titration+calorimetry+and+differential+scanning+calorimetry+as+complementary+tools+to+investigate+the+energetics+of+biomolecular+recognition.">Isothermal titration calorimetry and differential scanning calorimetry as complementary tools to investigate the energetics of biomolecular recognition.</a> J. Mol. Recognit. 12, 3-18 (1999).</li><li>Velazquez-Campoy, A., Leavitt, S. A., Freire, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+protein-protein+interactions+by+isothermal+titration+calorimetry.">Characterization of protein-protein interactions by isothermal titration calorimetry.</a> Methods Mol. Biol. 261, 35-54 (2004).</li><li>Turnbull, W. B., Daranas, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+value+of+c:+can+low+affinity+systems+be+studied+by+isothermal+titration+calorimetry.">On the value of c: can low affinity systems be studied by isothermal titration calorimetry.</a> J. Am. Chem. Soc. 125, 14859-14866 (2003).</li><li>Fukada, H., Takahashi, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enthalpy+and+heat+capacity+changes+for+the+proton+dissociation+of+various+buffer+components+in+0.1+M+potassium+chloride.">Enthalpy and heat capacity changes for the proton dissociation of various buffer components in 0.1 M potassium chloride.</a> Proteins. 33, 159-166 (1998).</li><li>Houtman, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studying+multisite+binary+and+ternary+protein+interactions+by+global+analysis+of+isothermal+titration+calorimetry+data+in+SEDPHAT:+application+to+adaptor+protein+complexes+in+cell+signaling.">Studying multisite binary and ternary protein interactions by global analysis of isothermal titration calorimetry data in SEDPHAT: application to adaptor protein complexes in cell signaling.</a> Protein Sci. 16, 30-42 (2007).</li><li>Bradrick, T. D., Beechem, J. M., Howell, E. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unusual+binding+stoichiometries+and+cooperativity+are+observed+during+binary+and+ternary+complex+formation+in+the+single+active+pore+of+R67+dihydrofolate+reductase,+a+D2+symmetric+protein.">Unusual binding stoichiometries and cooperativity are observed during binary and ternary complex formation in the single active pore of R67 dihydrofolate reductase, a D2 symmetric protein.</a> Biochemistry. 35, 11414-11424 (1996).</li><li>Shenoy, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multisite+and+multivalent+binding+between+cyanovirin-N+and+branched+oligomannosides:+calorimetric+and+NMR+characterization.">Multisite and multivalent binding between cyanovirin-N and branched oligomannosides: calorimetric and NMR characterization.</a> Chem. Biol. 9, 1109-1118 (2002).</li><li>Chopra, S., Lynch, R., Kim, S. H., Jackson, M., Howell, E. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+temperature+and+viscosity+on+R67+dihydrofolate+reductase+catalysis.">Effects of temperature and viscosity on R67 dihydrofolate reductase catalysis.</a> Biochemistry. 45, 6596-6605 (2006).</li><li>Norris, A. L., Serpersu, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interactions+of+Coenzyme+A+with+the+Aminoglycoside+Acetyltransferase+(3)-IIIb+and+Thermodynamics+of+a+Ternary+System.">Interactions of Coenzyme A with the Aminoglycoside Acetyltransferase (3)-IIIb and Thermodynamics of a Ternary System.</a> Biochemistry. 49, 4036-4042 (2010).</li><li>Falconer, R. J., Penkova, A., Jelesarov, I., Collins, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Survey+of+the+year+2008:+applications+of+isothermal+titration+calorimetry.">Survey of the year 2008: applications of isothermal titration calorimetry.</a> J. Mol. Recognit. 23, 395-413 (2010).</li><li>Ababou, A., Ladbury, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Survey+of+the+year+2005:+literature+on+applications+of+isothermal+titration+calorimetry.">Survey of the year 2005: literature on applications of isothermal titration calorimetry.</a> J. Mol. Recognit. 20, 4-14 (2007).</li><li>Buurma, N. J., Haq, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+the+analysis+of+isothermal+titration+calorimetry+data+for+ligand-DNA+interactions.">Advances in the analysis of isothermal titration calorimetry data for ligand-DNA interactions.</a> Methods. 42, 162-172 (2007).</li><li>Salim, N. N., Feig, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isothermal+titration+calorimetry+of+RNA.">Isothermal titration calorimetry of RNA.</a> Methods. 47, 198-205 (2009).</li><li>De, M., You, C. C., Srivastava, S., Rotello, V. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomimetic+interactions+of+proteins+with+functionalized+nanoparticles:+a+thermodynamic+study.">Biomimetic interactions of proteins with functionalized nanoparticles: a thermodynamic study.</a> J. Am. Chem. Soc. 129, 10747-10753 (2007).</li><li>Velazquez-Campoy, A., Freire, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isothermal+titration+calorimetry+to+determine+association+constants+for+high-affinity+ligands.">Isothermal titration calorimetry to determine association constants for high-affinity ligands.</a> Nat. Protoc. 1, 186-191 (2006).</li><li>Velazquez Campoy, A., Freire, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ITC+in+the+post-genomic+era...?+Priceless.">ITC in the post-genomic era...? Priceless.</a> Biophys. Chem. 115, 115-124 (2005).</li><li>Harries, D., Rau, D. C., Parsegian, V. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Solutes+probe+hydration+in+specific+association+of+cyclodextrin+and+adamantane.">Solutes probe hydration in specific association of cyclodextrin and adamantane.</a> J. Am. Chem. Soc. 127, 2184-2190 (2005).</li><li>Chervenak, M. C., Toone, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+direct+measure+of+the+contribution+of+solvent+reorganization+to+the+enthalpy+of+binding.">A direct measure of the contribution of solvent reorganization to the enthalpy of binding.</a> J. Am. Chem. Soc. 116, 10533-10539 (1994).</li><li>Taneva, S. G., Moro, F., Velazquez-Campoy, A., Muga, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Energetics+of+nucleotide-induced+DnaK+conformational+states.">Energetics of nucleotide-induced DnaK conformational states.</a> Biochemistry. 49, 1338-1345 (2010).</li><li>Jelesarov, I., Bosshard, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thermodynamics+of+ferredoxin+binding+to+ferredoxin:NADP++reductase+and+the+role+of+water+at+the+complex+interface.">Thermodynamics of ferredoxin binding to ferredoxin:NADP+ reductase and the role of water at the complex interface.</a> Biochemistry. 33, 13321-13328 (1994).</li><li>Burrows, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+the+monomer-dimer+equilibrium+of+interleukin-8+reveals+it+is+a+monomer+at+physiological+concentration.">Determination of the monomer-dimer equilibrium of interleukin-8 reveals it is a monomer at physiological concentration.</a> Biochemistry. 33, 12741-12745 (1994).</li><li>Baker, B. M., Murphy, K. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+linked+protonation+effects+in+protein+binding+reactions+using+isothermal+titration+calorimetry.">Evaluation of linked protonation effects in protein binding reactions using isothermal titration calorimetry.</a> Biophys. J. 71, 2049-2055 (1996).</li></ol>

没有利益冲突的声明。

ITC已广泛用于蛋白配体，12 DNA配体13和RNA 大分子的14项研究的研究与学习的配体大分子的相互作用，11。 ITC甚至可以运行，即形成均匀的悬浮固体材料，如纳米粒子。15此外，三元体系，其中一个配体已经绑定到高分子和滴定第二配体，可以被用来确定了热力学，例如，基板具有约束力一种酶的辅助因子的系统。研究还可以进行具有很高的亲和力，通常会超过执行竞争较弱的具有约束力的配体结合分析国际贸易中心的检测限为分子16。弱约束力的信息配体可以通过竞争分析17约束作用的水可以通过探索，随着溶剂重组焓的依赖国际贸易中心，18 19最近，DnaK的变种构象变化的热力学测定后绑定ADP和ATP，可以由国贸中心 20蛋白的蛋白质协会研究，产生异质复合物 21以及HOMO关联的信息 。22的结合焓温度效应会给绑定事件的热容量2的数量结合后吸收或释放的质子，也可以从不同的电离焓的缓冲区进行实验来确定。5，如果更多的国际贸易中心的研究是在不同pH值，那么有可能确定所涉及组的pKa 23。 总之，精确的测量大分子与配体的浓度是一个良好的国际贸易中心实验来说是必要的。样品浓度也需要一个适当的范围内内，以获得可靠的数据。护理应采取的缓冲区，小分子杂质和pH值不匹配，会造成在热谱文物。

<table border="1"><tbody><tr><th>试剂名称</th><th>公司</th><th>目录编号</th></tr><tr><td> 1.7毫升离心管</td><td> Fisher Scientific则</td><td> 02-681-282 </td></tr><tr><td>猎鹰管15毫升</td><td> Fisher Scientific则</td><td> 14 - 959 - 49B </td></tr><tr><td>汉密尔顿注射器2.5毫升</td><td> MICROCAL </td><td> SYN161714 </td></tr></tbody></table>

isothermal titration calorimetry for measuring macromolecule-ligand affinity

等温滴定量热（ITC）是一个有用的工具，为了解一项具有约束力的反应的完整的热力学图片。在生物科学，大分子的相互作用是理解细胞的机制至关重要。实验条件，如缓冲区和温度，可以根据特定约束力的制度，正在研究。然而，细心的规划是必要的，因为某些配体和高分子浓度范围是必要的，以获取有用的数据。大分子与配体的浓度要准确可靠的结果，决定。护理也需要准备样品的杂质可以显著影响的实验时，应采取的。当ITC的实验与对照，正确执行的，有用的绑定信息，如化学计量学，亲和力和焓，得到。运行更多的试验，根据不同的缓冲区或温度条件下，可以得到更详细的信息，有关系统。一个协议是为一个国际贸易中心实验的基本格局。

Watch this Scientific Journal Video about Isothermal Titration Calorimetry for Measuring Macromolecule-Ligand Affinity at JoVE.com

Isothermal Titration Calorimetry for Measuring Macromolecule-Ligand Affinity

使用温和的亲和力，以监测生物系统的结合热力学等温滴定量热的一个协议。

用于测量高分子配体亲和等温滴定量热

Isothermal titration calorimetry (ITC) is a useful tool for understanding the complete thermodynamic picture of a binding reaction.  In biological ...

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Unit 1

Acid–Base Titration

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52.5K 次观看.  University of Tennessee. 使用温和的亲和力，以监测生物系统的结合热力学等温滴定量热的一个协议。

视频: Isothermal Titration Calorimetry for Measuring Macromolecule-Ligand Affinity

Titration of Human Coronaviruses Using an Immunoperoxidase Assay

Determination of infectious viral titers is a basic and essential experimental approach for virologists. Classical plaque assays cannot be used for viruses that do not cause significant cytopathic effects, which is the case for prototype strains 229E and OC43 of human coronavirus (HCoV). Therefore, an alternative indirect immunoperoxidase assay (IPA) was developed for the detection and titration of these viruses and is described herein. Susceptible cells are inoculated with serial logarithmic dilutions of virus-containing samples in a 96-well plate format. After viral growth, viral detection by IPA yields the infectious virus titer, expressed as 'Tissue Culture Infectious Dose 50 percent' (TCID50). This represents the dilution of a virus-containing sample at which half of a series of laboratory wells contain infectious replicating virus. This technique provides a reliable method for the titration of HCoV-229E and HCoV-OC43 in biological samples such as cells, tissues and fluids. This article is based on work first reported in Methods in Molecular Biology (2008) volume 454, pages 93-102.

In this video, we demonstrate an alternative method for detection and titering of viruses using an enzymatic antigen detection technique known as an immunoperoxidase assay. Here, we will show you how to collect your viral samples, prepare the cells for testing, and finally the immunoperoxidase assay using serial dilutions to determine the viral titer.

人类冠状病毒的滴定使用免疫过氧化物酶含量

Determination of infectious viral titers is a basic and essential experimental approach for virologists. Classical plaque assays cannot be used for ...

科研

生物学

Collecting Variable-concentration Isothermal Titration Calorimetry Datasets in Order to Determine Binding Mechanisms

Isothermal titration calorimetry (ITC) is commonly used to determine the thermodynamic parameters associated with the binding of a ligand to a host macromolecule. ITC has some advantages over common spectroscopic approaches for studying host/ligand interactions. For example, the heat released or absorbed when the two components interact is directly measured and does not require any exogenous reporters. Thus the binding enthalpy and the association constant (Ka) are directly obtained from ITC data, and can be used to compute the entropic contribution. Moreover, the shape of the isotherm is dependent on the c-value and the mechanistic model involved. The c-value is defined as c = n[P]tKa, where [P]t is the protein concentration, and n is the number of ligand binding sites within the host. In many cases, multiple binding sites for a given ligand are non-equivalent and ITC allows the characterization of the thermodynamic binding parameters for each individual binding site. This however requires that the correct binding model be used. This choice can be problematic if different models can fit the same experimental data. We have previously shown that this problem can be circumvented by performing experiments at several c-values. The multiple isotherms obtained at different c-values are fit simultaneously to separate models. The correct model is next identified based on the goodness of fit across the entire variable-c dataset. This process is applied here to the aminoglycoside resistance-causing enzyme aminoglycoside N-6'-acetyltransferase-Ii (AAC(6')-Ii). Although our methodology is applicable to any system, the necessity of this strategy is better demonstrated with a macromolecule-ligand system showing allostery or cooperativity, and when different binding models provide essentially identical fits to the same data. To our knowledge, there are no such systems commercially available. AAC(6')-Ii, is a homo-dimer containing two active sites, showing cooperativity between the two subunits. However ITC data obtained at a single c-value can be fit equally well to at least two different models a two-sets-of-sites independent model and a two-site sequential (cooperative) model. Through varying the c-value as explained above, it was established that the correct binding model for AAC(6')-Ii is a two-site sequential binding model. Herein, we describe the steps that must be taken when performing ITC experiments in order to obtain datasets suitable for variable-c analyses.

ITC is a powerful tool for studying the binding of a ligand to its host. In complex systems however, several models may fit the data equally well. The method described here provides a means to elucidate the appropriate binding model for complex systems and extract the corresponding thermodynamic parameters.

收集可变浓度等温滴定量热数据集，以确定约束机制

Isothermal titration calorimetry (ITC) is commonly used to determine the thermodynamic parameters associated with the binding of a ligand to a host ...

High-throughput Purification of Affinity-tagged Recombinant Proteins

X-ray crystallography is the method of choice for obtaining a detailed view of the structure of proteins. Such studies need to be complemented by further biochemical analyses to obtain detailed insights into structure/function relationships. Advances in oligonucleotide- and gene synthesis technology make large-scale mutagenesis strategies increasingly feasible, including the substitution of target residues by all 19 other amino acids. Gain- or loss-of-function phenotypes then allow systematic conclusions to be drawn, such as the contribution of particular residues to catalytic activity, protein stability and/or protein-protein interaction specificity.
In order to attribute the different phenotypes to the nature of the mutation - rather than to fluctuating experimental conditions - it is vital to purify and analyse the proteins in a controlled and reproducible manner. High-throughput strategies and the automation of manual protocols on robotic liquid-handling platforms have created opportunities to perform such complex molecular biological procedures with little human intervention and minimal error rates1-5.
Here, we present a general method for the purification of His-tagged recombinant proteins in a high-throughput manner. In a recent study, we applied this method to a detailed structure-function investigation of TFIIB, a component of the basal transcription machinery. TFIIB is indispensable for promoter-directed transcription in vitro and is essential for the recruitment of RNA polymerase into a preinitiation complex6-8. TFIIB contains a flexible linker domain that penetrates the active site cleft of RNA polymerase9-11. This linker domain confers two biochemically quantifiable activities on TFIIB, namely (i) the stimulation of the catalytic activity during the 'abortive' stage of transcript initiation, and (ii) an additional contribution to the specific recruitment of RNA polymerase into the preinitiation complex4,5,12 . We exploited the high-throughput purification method to generate single, double and triple substitution and deletions mutations within the TFIIB linker and to subsequently analyse them in functional assays for their stimulation effect on the catalytic activity of RNA polymerase4. Altogether, we generated, purified and analysed 381 mutants - a task which would have been time-consuming and laborious to perform manually. We produced and assayed the proteins in multiplicates which allowed us to appreciate any experimental variations and gave us a clear idea of the reproducibility of our results.
This method serves as a generic protocol for the purification of His-tagged proteins and has been successfully used to purify other recombinant proteins. It is currently optimised for the purification of 24 proteins but can be adapted to purify up to 96 proteins.

We describe a method for the affinity-tagged purification of recombinant proteins using liquid-handling robotics. This method is generally applicable to the small-scale purification of soluble His-tagged proteins in a high-throughput format.

高通量的亲和标签的重组蛋白的纯化

X-ray crystallography is the method of choice for obtaining a detailed view of the structure of proteins. Such studies need to be complemented by further ...

Biochemical Titration of Glycogen In vitro

Glycogen is the main energetic polymer of glucose in vertebrate animals and plays a crucial role in whole body metabolism as well as in cellular metabolism. Many methods to detect glycogen already exist but only a few are quantitative. We describe here a method using the Abcam Glycogen assay kit, which is based on specific degradation of glycogen to glucose by glucoamylase. Glucose is then specifically oxidized to a product that reacts with the OxiRed probe to produce fluorescence. Titration is accurate, sensitive and can be achieved on cell extracts or tissue sections. However, in contrast to other techniques, it does not give information about the distribution of glycogen in the cell. As an example of this technique, we describe here the titration of glycogen in two cell lines, Chinese hamster lung fibroblast CCL39 and human colon carcinoma LS174, incubated in normoxia (21% O2) versus hypoxia (1% O2). We hypothesized that hypoxia is a signal that prepares cells to synthesize and store glycogen in order to survive1.

Biochemical Titration of Glycogen In vitro

We describe here an accurate, reproducible and convenient biochemical method for titration of glycogen in vitro. This technique uses the Abcam Glycogen assay kit and is based on successive hydrolysis of glycogen to glucose and glucose titration by fluorescence.

糖原生化滴定<em&gt;在体外</em

Glycogen is the main energetic polymer of glucose in vertebrate animals and plays a crucial role in whole body metabolism as well as in cellular ...

A Protocol for Phage Display and Affinity Selection Using Recombinant Protein Baits

Using recombinant phage as a scaffold to present various protein portions encoded by a directionally cloned cDNA library to immobilized bait molecules is an efficient means to discover interactions. The technique has largely been used to discover protein-protein interactions but the bait molecule to be challenged need not be restricted to proteins. The protocol presented here has been optimized to allow a modest number of baits to be screened in replicates to maximize the identification of independent clones presenting the same protein. This permits greater confidence that interacting proteins identified are legitimate interactors of the bait molecule. Monitoring the phage titer after each affinity selection round provides information on how the affinity selection is progressing as well as on the efficacy of negative controls. One means of titering the phage, and how and what to prepare in advance to allow this process to progress as efficiently as possible, is presented. Attributes of amplicons retrieved following isolation of independent plaque are highlighted that can be used to ascertain how well the affinity selection has progressed. Trouble shooting techniques to minimize false positives or to bypass persistently recovered phage are explained. Means of reducing viral contamination flare up are discussed.

Phage display is a powerful technique to capture proteins or protein moieties that interact with an immobilized molecule of interest. Once a decision of the type of phage cDNA library to create and screen has been made, the protocol described here permits efficient affinity selection leading to identification of interactors.

一个协议，用于噬菌体展示和亲和选择以重组蛋白诱饵

Using  recombinant phage as a scaffold to present various protein portions encoded by  a directionally cloned cDNA library to immobilized bait molecules ...

Affinity-based Isolation of Tagged Nuclei from Drosophila Tissues for Gene Expression Analysis

Drosophila melanogaster embryonic and larval tissues often contain a highly heterogeneous mixture of cell types, which can complicate the analysis of gene expression in these tissues. Thus, to analyze cell-specific gene expression profiles from Drosophila tissues, it may be necessary to isolate specific cell types with high purity and at sufficient yields for downstream applications such as transcriptional profiling and chromatin immunoprecipitation. However, the irregular cellular morphology in tissues such as the central nervous system, coupled with the rare population of specific cell types in these tissues, can pose challenges for traditional methods of cell isolation such as laser microdissection and fluorescence-activated cell sorting (FACS). Here, an alternative approach to characterizing cell-specific gene expression profiles using affinity-based isolation of tagged nuclei, rather than whole cells, is described. Nuclei in the specific cell type of interest are genetically labeled with a nuclear envelope-localized EGFP tag using the Gal4/UAS binary expression system. These EGFP-tagged nuclei can be isolated using antibodies against GFP that are coupled to magnetic beads. The approach described in this protocol enables consistent isolation of nuclei from specific cell types in the Drosophila larval central nervous system at high purity and at sufficient levels for expression analysis, even when these cell types comprise less than 2% of the total cell population in the tissue. This approach can be used to isolate nuclei from a wide variety of Drosophila embryonic and larval cell types using specific Gal4 drivers, and may be useful for isolating nuclei from cell types that are not suitable for FACS or laser microdissection.

Affinity-based Isolation of Tagged Nuclei from Drosophila Tissues for Gene Expression Analysis

Drosophila tissues often contain a heterogeneous mixture of cell types.&#160;To examine gene expression in specific cell types from a particular tissue, nuclei can be genetically tagged and subsequently isolated using an affinity-based approach. Isolated nuclei can be used for downstream applications such as gene expression analysis and chromatin immunoprecipitation.

标记的细胞核从亲和力为基础的隔离<em&gt;果蝇</em&gt;组织的基因表达分析

Drosophila melanogaster embryonic and larval tissues often contain a highly heterogeneous mixture of cell types, which can complicate the analysis of gene ...

High-throughput Titration of Luciferase-expressing Recombinant Viruses

Standard plaque assays to determine infectious viral titers can be time consuming, are not amenable to a high volume of samples, and cannot be done with viruses that do not form plaques. As an alternative to plaque assays, we have developed a high-throughput titration method that allows for the simultaneous titration of a high volume of samples in a single day. This approach involves infection of the samples with a Firefly luciferase tagged virus, transfer of the infected samples onto an appropriate permissive cell line, subsequent addition of luciferin, reading of plates in order to obtain luminescence readings, and finally the conversion from luminescence to viral titers. The assessment of cytotoxicity using a metabolic viability dye can be easily incorporated in the workflow in parallel and provide valuable information in the context of a drug screen. This technique provides a reliable, high-throughput method to determine viral titers as an alternative to a standard plaque assay.

This article presents a high-throughput luciferase expression-based method of titrating various RNA and DNA viruses using automated and manual liquid handlers.

萤光素酶表达的重组病毒的高通量滴定

Standard plaque assays to determine infectious viral titers can be time consuming, are not amenable to a high volume of samples, and cannot be done with ...

Purification of Native Complexes for Structural Study Using a Tandem Affinity Tag Method

Affinity purification approaches have been successful in isolating native complexes for proteomic characterization. Structural heterogeneity and a degree of compositional heterogeneity of a complex do not usually impede progress in conducting such studies. In contrast, a complex intended for structural characterization should be purified in a state that is both compositionally and structurally homogeneous as well as at a higher concentration than required for proteomics. Recently, there have been significant advances in the application of electron microscopy for structure determination of large macromolecular complexes. This has heightened interest in approaches to purify native complexes of sufficient quality and quantity for structural determination by electron microscopy. The Tandem Affinity Purification (TAP) method has been optimized to extract and purify an 18-subunit, ~ 0.8 MDa ribonucleoprotein assembly from budding yeast (Saccharomyces cerevisiae) suitable for negative stain and electron cryo microscopy. Herein is detailed the modifications made to the TAP method, the rationale for making these changes, and the approaches taken to assay for a compositionally and structurally homogeneous complex.

The Tandem Affinity Purification (TAP) method has been used extensively to isolate native complexes from cellular extract, primarily eukaryotic, for proteomics. Here, we present a TAP method protocol optimized for purification of native complexes for structural studies.

本机配合物的净化对结构研究使用串联亲和标签法

Affinity purification approaches have been successful in isolating native complexes for proteomic characterization. Structural heterogeneity and a degree ...

A Customizable Chamber for Measuring Cell Migration

Cell migration is a vital part of immune responses, growth, and wound healing. Cell migration is a complex process that involves interactions between cells, the extracellular matrix, and soluble and non-soluble chemical factors (e.g., chemoattractants). Standard methods for measuring the migration of cells, such as the Boyden chamber assay, work by counting cells on either side of a divider. These techniques are easy to use; however, they offer little geometric modification for different applications. In contrast, microfluidic devices can be used to observe cell migration with customizable concentration gradients of soluble factors1,2. However, methods for making microfluidics based assays can be difficult to learn.
Here, we describe an easy method for creating cell culture chambers to measure cell migration in response to chemical concentration gradients. Our cell migration chamber method can create different linear concentration gradients in order to study cell migration for a variety of applications. This method is relatively easy to use and is typically performed by undergraduate students.
The microchannel chamber was created by placing an acrylic insert in the shape of the final microchannel chamber well into a Petri dish. After this, poly(dimethylsiloxane) (PDMS) was poured on top of the insert. The PDMS was allowed to harden and then the insert was removed. This allowed for the creation of wells in any desired shape or size. Cells may be subsequently added to the microchannel chamber, and soluble agents can be added to one of the wells by soaking an agarose block in the desired agent. The agarose block is added to one of the wells, and time-lapse images can be taken of the microchannel chamber in order to quantify cell migration. Variations to this method can be made for a given application, making this method highly customizable.

This protocol details a customizable method to measure cell migration in response to chemoattractants that may also be used to determine the diffusion rate of a drug out of a polymer matrix.

定制室测定细胞迁移

Cell migration is a vital part of immune responses, growth, and wound healing. Cell migration is a complex process that involves interactions between ...

Simultaneous Affinity Enrichment of Two Post-Translational Modifications for Quantification and Site Localization

Studying multiple post-translational modifications (PTMs) of proteins is a crucial step to understand PTM crosstalk and gain more holistic insights into protein function. Despite the importance of multi-PTM enrichment studies, few studies investigate more than one PTM at a time, due partially to the expenses, time, and large protein quantities required to perform multiple global proteomic analysis of PTMs. The &#34;one-pot&#34; affinity enrichment detailed in this protocol overcomes these barriers by permitting the simultaneous identification and quantification of peptides with lysine residues containing acetylation and succinylation PTMs with low amounts of sample input. The protocol involves preparation of protein lysate from mouse livers of SIRT5 knockout mice, performance of trypsin digestion, enrichment for PTMs, and performance of mass spectrometric analysis using a data-independent acquisition (DIA) workflow. Because this workflow allows for the enrichment of two PTMs from the same sample simultaneously, it provides a practical tool to study PTM crosstalk without requiring large amounts of samples, and it greatly reduces the time required for sample preparation, data acquisition, and analysis. The DIA component of the workflow provides comprehensive PTM-specific information. This is particularly important when studying PTM site localization, as DIA provides comprehensive sets of fragment ions that can be computationally deciphered to differentiate between different PTM localization isoforms.

This workflow describes the performance of time- and cost-efficient enrichment of multiple protein post-translational modifications (PTMs) simultaneously for quantitative global proteomic analysis. The protocol utilizes peptide-level PTM enrichment with multiple conjugated antibodies, followed by data-independent acquisition mass spectrometry analysis to gain biological insights into PTM crosstalk.

量化和站点本地化两个翻译后修改的同步亲和力丰富

Studying multiple post-translational modifications (PTMs) of proteins is a crucial step to understand PTM crosstalk and gain more holistic insights into ...