Name: differential scanning calorimetry — a method for assessing the thermal stability and conformation of protein antigen
Uploaded: 2017-03-04T13:00:00.000+00:00
Duration: 8 min 13 s
Description: Watch this Scientific Journal Video about Differential Scanning Calorimetry â€” A Method for Assessing the Thermal Stability and Conformation of Protein Antigen at JoVE.com

作者非常感谢约瑟夫曼奇尼（原GE医疗集团），帕维尔Czudec，托马斯·凯奇（马尔文仪器有限公司）为他们的差示扫描量热，Sasmit德希穆克和韦伯斯特Magcalas他们讨论安装和培训的作用。

1.仪器启动<ol><li>接通差示扫描量热计并增加压力在细胞中抑制样品的沸点以及防止形成气泡在升高的温度。这通常是通过供给氮气入系统来实现的。 </li><li>取决于细胞（ 例如 ，钽，金，铂等 ）的构成材料，根据制造商建议的压力，以避免损坏电池调节氮气供给的压力。例如，设置氮气供给到45磅为用于开发本程序的仪器的压力，并且压力高于80磅可能会损坏电池。 </li><li>确保所有的清洁剂储填充到所需体积。所需的清洁剂包括洗涤剂和水清洗和每个样本运行后分别清洗细胞。 </li><li>设置采样保持室的温度到一个适当的值，优选为5℃，保持试验前的样品的完整性。 </li></ol> 2.样品制备<ol><li>透析针对将被用作实验中的参考缓冲器中的样本。可替代地，可以使用在蛋白质纯化（ 即柱洗脱）的最终步骤中收集的洗脱缓冲液。 </li><li>确定使用最合适的蛋白浓度的测定方法的蛋白质样品的浓度如Kjedahl方法33或Lowry法34。对于结果的客观比较，均使用同一研究中同样的方法。所需的浓度范围可根据仪器的型号而改变。在此协议中使用的仪器，优选的工作范围是0.5 - 1毫克/毫升。 </li><li>德加在真空中的样品和参考缓冲器，以摆脱可能会导致体积inaccur微泡ACY。此步骤可以跳过对更新量热模型。 </li><li>在层流生物控制柜使用微量和无菌针头，加载样品和它们各自的缓冲器中对插入96与仪器兼容孔板中。填充缓冲器中的前两对孔中，最后两对水的缓冲器缓冲和分别水扫描。缓冲缓冲扫描验证样品测量（ 即仪器的误差评估），以及建立一个基线前仪器的适用性;而水的扫描被执行清洗细胞。 </li><li>盖用密封膜中的96孔板，并确保该井正在服用的板出来的生物安全柜中，以避免样品污染之前正确密封。 </li><li>放置板在合适的方向的试样收容室中。 </li></ol> 3.实验参数设置注:根据我nstrumentation，样品可以使用手动注射器，或自动地使用自动进样器被加载到细胞内。在这种情况下（ 即一个工业设置），自动进样器用于以节省时间。 <ol><li>使用采集软件，请在板加载按2.4节的顺序样本信息。如果有输入的浓度，否则输入的浓度值到数据分析（4.2节）之前的分析软件。 </li><li>选择，以确保用洗涤剂细胞清洗每样品扫描，其应遵循由多个水冲洗步骤，以确保没有洗涤剂残余物留在细胞之前的选项。 </li><li>设置实验至20℃的起始温度，但是这可以根据样品的先验知识而变化。对于公知的蛋白质，预先确定的开始温度都可以使用，而可应用于未知样品更低的起始温度。 </li><li>设置最终温度实验的， 例如 100℃。最终温度可以根据样品的先验知识而变化。 </li><li>设置实验的扫描速率， 例如 60℃/小时，这是典型的扫描速率。然而，扫描速度可根据样品的现有知识不同， 例如在 90℃/小时，或120℃/小时。这是明智的扫描不同扫描速度未知样品评估展开的动力学。 </li><li>重新扫描样本调查热解折叠的可逆性。展开的蛋白质被认为是可逆如果对于第二扫描中获得的焓为第一扫描的焓值的至少80％。 </li><li>设置-实验后调温至10℃，以保持热量的细胞的完整性。 </li><li>验证实验的设置参数是执行实验前正确的。如果一切就绪，开始实验。 </li></ol> 4.数据的alysis <ol><li>从实验中检索原始数据，并在同一时间进行分析选择一个样本。减法参考扫描， 即缓冲液，从样品扫描。 注:参考减法通过DSC仪器的较新型号自动地进行。 </li><li>如果它被省略每节3.1输入样品浓度值。 </li><li>适合并从所获取的温谱图中减去基线以考虑在由疏水性基团的曝光时展开造成水的蛋白质的折叠和非折叠状态的热容量的差异。直链或三次曲线拟合可以根据该示例的DSC分布图的形状被应用。为了保持一致性，相同类型的接头有一个研究中， 例如，实时稳定性研究过程中使用。此步骤需要处理的曲线峰积分，以获得过渡的焓。 </li><li>使用非线性最小二乘拟合进行峰积分。根据产品知识应用于两个国家还是非二态模型。二态模型可以用于单合作热转变，而对于未知的蛋白质，适用于非二态模型直到进一步的产品知识是可用的。如果适用，调整曲线，使用设备的软件的迭代曲线拟合函数，直到卡方值保持不变拟合。 </li><li>得到的结果将显示转变温度（Tm）为，量热焓（ΔH），和样品的范特霍夫焓（ΔHV H）的中点值。 </li></ol>

<ol>
	<li>Gill, P., Moghadam, T. T., Ranjbar, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+scanning+calorimetry+techniques:+applications+in+biology+and+nanoscience.">Differential scanning calorimetry techniques: applications in biology and nanoscience.</a> J. Biomol. Tech. 21 (4), 167-193 (2010).</li><li>Watson, E. S., O'Neil, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+microcalorimeter.">Differential microcalorimeter.</a> Patent. , (1966).</li><li>O'Neill, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Analysis+of+a+Temperature-Controlled+Scanning+Calorimeter.">The Analysis of a Temperature-Controlled Scanning Calorimeter.</a> Anal. Chem. 36 (7), 1238-1245 (1964).</li><li>O'Neil, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+Specific+Heat+Functions+by+Differential+Scanning+Calorimetry.">Measurement of Specific Heat Functions by Differential Scanning Calorimetry.</a> Anal. Chem. 38 (10), 1331-1336 (1966).</li><li>Privalov, P. L., Monaselidze, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Mol. Biol. 6, 7-33 (1975).</li><li>Privalov, P. L., Plotnikov, V. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adiabatic+differential+microcalorimeter.">Adiabatic differential microcalorimeter.</a> &#1040;&#1076;&#1080;&#1072;&#1073;&#1072;&#1090;&#1080;&#1095;&#1077;&#1089;&#1082;&#1080;&#1081; &#1076;&#1080;&#1092;&#1092;&#1077;&#1088;&#1077;&#1085;&#1094;&#1080;&#1072;&#1083;&#1100;&#1085;&#1099;&#1081; &#1084;&#1080;&#1082;&#1088;&#1086;&#1082;&#1072;&#1083;&#1086;&#1088;&#1080;&#1084;&#1077;&#1090;&#1088;. , (1970).</li><li>Andronikashvili, E. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calorimetric+study+of+the+nature+of+the+intramolecular+fusion+of+collagen,+isolated+from+transplantable+tumor.">Calorimetric study of the nature of the intramolecular fusion of collagen, isolated from transplantable tumor.</a> Dokl. Akad. Nauk. 183 (1), 212-214 (1968).</li><li>Andronikashvili, E. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Conformational Changes of Biopolymers in Solution. , 171-173 (1973).</li><li>Andronikashvili, E. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Malignant+transformation+and+changes+in+various+physic-chemical+properties+of+macromolecules+and+supramolecular+structures.">Malignant transformation and changes in various physic-chemical properties of macromolecules and supramolecular structures.</a> Biofizika. 32 (5), 782-799 (1987).</li><li>Velicelebi, G., Sturtevant, J. M. <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+the+denaturation+of+lysozyme+in+alcohol-water+mixtures.">Thermodynamics of the denaturation of lysozyme in alcohol-water mixtures.</a> Biochemistry. 18 (7), 1180-1186 (1979).</li><li>Sturtevant, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heat+capacity+and+entropy+changes+in+processes+involving+proteins.">Heat capacity and entropy changes in processes involving proteins.</a> Proc. Natl. Acad. Sci. 74 (6), 2236-2240 (1977).</li><li>Sturtevant, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biochemical+applications+of+differential+scanning+calorimetry.">Biochemical applications of differential scanning calorimetry.</a> Annu. Rev. of Phys. Chem. 38, 463-488 (1987).</li><li>Jackson, W. M., Brandts, J. F. <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+protein+denaturation.+A+calorimetric+study+of+the+reversible+denaturation+of+chymotrypsinogen+and+conclusions+regarding+the+accuracy+of+the+two-state+approximation.">Thermodynamics of protein denaturation. A calorimetric study of the reversible denaturation of chymotrypsinogen and conclusions regarding the accuracy of the two-state approximation.</a> Biochem. 9 (11), 2294-2301 (1970).</li><li>Privalov, P. L., Khechinashvili, N. N., Atanasov, B. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thermodynamic+analysis+of+thermal+transitions+in+globular+proteins.+I.+Calorimetric+study+of+chymotrypsinogen,+ribonuclease+and+myoglobin.">Thermodynamic analysis of thermal transitions in globular proteins. I. Calorimetric study of chymotrypsinogen, ribonuclease and myoglobin.</a> Biopolymers. 10 (10), 1865-1890 (1971).</li><li>Privalov, P. L., Khechinashvili, N. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+thermodynamic+approach+to+the+problem+of+stabilization+of+globular+protein+structure:+a+calorimetric+study.">A thermodynamic approach to the problem of stabilization of globular protein structure: a calorimetric study.</a> J. Mol. Biol. 86 (3), 665-684 (1974).</li><li>Privalov, P. L., Potekhin, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scanning+microcalorimetry+in+studying+temperature-induced+changes+in+proteins.">Scanning microcalorimetry in studying temperature-induced changes in proteins.</a> Methods Enzymol. 131, 4-51 (1986).</li><li>Veprintseva, O. D., Emelyanenko, V. I., Konstantinova, V. V., Shnyrov, V. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+importance+of+structural+changes+in+bacteriophage+T4+tail+proteins.">The importance of structural changes in bacteriophage T4 tail proteins.</a> Biofizika. 33, 954-961 (1988).</li><li>Shutilova, N., Semenova, G., Klimov, V., Shnyrov, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temperature-induced+functional+and+structural+transformations+of+the+photosystem+II+oxygen-evolving+complex+in+spinach+subchloroplast+preparations.">Temperature-induced functional and structural transformations of the photosystem II oxygen-evolving complex in spinach subchloroplast preparations.</a> Biochem. Mol. Biol. Int. 35 (6), 1233-1243 (1995).</li><li>Sanina, N. M., Kostetsky, E. Y. a., Shnyrov, V. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calorimetric+investigation+of+phosphatidyl+choline+from+membranes+of+marine+invertebrates.">Calorimetric investigation of phosphatidyl choline from membranes of marine invertebrates.</a> J. Evol. Biokhim. Physiol. 23, 451-460 (1987).</li><li>Oreshkin, E. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conformational+changes+in+the+muscle+proteins+of+cured+beef+during+heating.">Conformational changes in the muscle proteins of cured beef during heating.</a> Meat Science. 16 (4), 297-305 (1986).</li><li>Kirkitadze, M., Hu, J., Tang, M., Carpick, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Qualification+of+a+differential+scanning+calorimetry+method+for+biophysical+characterization+of+monoclonal+antibodies+and+protein+vaccine+antigens.">Qualification of a differential scanning calorimetry method for biophysical characterization of monoclonal antibodies and protein vaccine antigens.</a> Pharm. Bioprocess. 2 (6), 491-498 (2014).</li><li>Jiskoot, W., Daan, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Methods for structural analysis of protein pharmaceuticals. 3, (2005).</li><li>Plotnikov, V. V., Brandts, M., Lin, L. N., Brandts, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+ultrasensitive+scanning+calorimeter.">A new ultrasensitive scanning calorimeter.</a> Anal. Biochem. 250 (2), 237-244 (1997).</li><li>Plotnikov, V. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+autosampling+differential+scanning+calorimeter+instrument+for+studying+molecular+interactions.">An autosampling differential scanning calorimeter instrument for studying molecular interactions.</a> Assay Drug Dev. Technol. 1 (1), 83-90 (2002).</li><li>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=Differential+scanning+calorimetry.">Differential scanning calorimetry.</a> Protein Stability and Folding: Theory and Practice. Method in Molecular Biology. 40, 191-218 (1995).</li><li>Freire, E., van Osdol, W. W., Mayorga, O. L., Sanchez-Ruiz, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calorimetrically+determined+dynamics+of+complex+unfolding+transitions+in+proteins.">Calorimetrically determined dynamics of complex unfolding transitions in proteins.</a> Annu. Rev. Biophys. Biophys. Chem. 19, 159-188 (1990).</li><li>Johnson, C. R., Morin, P. E., Arrowsmith, C. H., 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=Thermodynamic+analysis+of+the+structural+stability+of+the+tetrameric+oligomerization+domain+of+p53+tumor+suppressor.">Thermodynamic analysis of the structural stability of the tetrameric oligomerization domain of p53 tumor suppressor.</a> Biochemistry. 34 (16), 5309-5316 (1995).</li><li>Kasimova, M. R., Sam, J. M., 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=The+conformational+equilibrium+of+human+growth+hormone.">The conformational equilibrium of human growth hormone.</a> J. Mol. Biol. 277 (2), 409-418 (1998).</li><li>Saboury, A. A., Moosavi-Movahedi, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clarification+of+calorimetric+and+van't+hoff+enthalpies+for+evaluation+of+protein+transition+states.">Clarification of calorimetric and van't hoff enthalpies for evaluation of protein transition states.</a> Biochem Edu. 22 (4), 210-211 (1994).</li><li>Privalov, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microcalorimetry+of+proteins+and+their+complexes.">Microcalorimetry of proteins and their complexes.</a> Protein Structure, Stability, and Interactions. Method in Molecular Biology. 490, 1-39 (2009).</li><li>Dehghan-Nayeri, N., Rezaei-Tavirani, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Interpretation+of+Protein+Structure+Through+Relationship+of+Melting+Point+(Tm)+and+Enthalpy+of+Unfolding+(&#916;HU).">The Interpretation of Protein Structure Through Relationship of Melting Point (Tm) and Enthalpy of Unfolding (&#916;HU).</a> Int J Anal Pharma Biomed Sci. 4 (1), 47-50 (2015).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stability+testing+of+new+drug+substances+and+products.">Stability testing of new drug substances and products.</a> Guideline, ICH Harmonised Tripartite. 1 (2), (2003).</li><li>Kjeldahl, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neue+Methode+zur+Bestimmung+des+Stickstoffs+in+organischen+K&#246;rpern+(New+method+for+the+determination+of+nitrogen+in+organic+substances).">Neue Methode zur Bestimmung des Stickstoffs in organischen K&#246;rpern (New method for the determination of nitrogen in organic substances).</a> Zeitschrift f&#252;r analytische Chemie. 22 (1), 366-383 (1883).</li><li>Lowry, O. H., Rosebrough, N. J., Farr, A. L., Randall, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+measurement+with+the+Folin+phenol+reagent.">Protein measurement with the Folin phenol reagent.</a> J. Biol. Chem. 193 (1), 265-275 (1951).</li><li>H&#214;hne, G., Hemminger, W., Flammersheim, H. 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> Differential Scanning Calorimetry. , (2003).</li><li>Porter, L. L., George, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+thermodynamic+definition+of+protein+domains.">A thermodynamic definition of protein domains.</a> Proc. Natl. Acad. Sci. USA. 109 (24), 9420-9425 (2012).</li><li>Frokjaer, S., Daniel, E. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+drug+stability:+a+formulation+challenge.">Protein drug stability: a formulation challenge.</a> Nature Reviews Drug Discovery. 4, 298-306 (2005).</li><li>Johnson, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+scanning+calorimetry+as+a+tool+for+protein+folding+and+stability.">Differential scanning calorimetry as a tool for protein folding and stability.</a> Arch. Biochem. Biophys. 531, 100-109 (2013).</li><li>Chiu, M. H., Elmar, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+scanning+calorimetry:+an+invaluable+tool+for+a+detailed+thermodynamic+characterization+of+macromolecules+and+their+interactions.">Differential scanning calorimetry: an invaluable tool for a detailed thermodynamic characterization of macromolecules and their interactions.</a> J. Pharm. Bioallied Sci. 3 (1), 39-59 (2011).</li><li>Hirst, J. D., Charles, L. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Helicity,+circular+dichroism+and+molecular+dynamics+of+proteins.">Helicity, circular dichroism and molecular dynamics of proteins.</a> J. Mol. Biol. 243 (2), 173-178 (1994).</li><li>Kirkitadze, M. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Central+modules+of+the+vaccinia+virus+complement+control+protein+are+not+in+extensive+contact.">Central modules of the vaccinia virus complement control protein are not in extensive contact.</a> Biochem. J. 344 (1), 167-175 (1999).</li><li>Freire, E., Sch&#214;n, A., Hutchins, B. M., Brown, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+denaturation+as+a+tool+in+the+formulation+optimization+of+biologics.">Chemical denaturation as a tool in the formulation optimization of biologics.</a> Drug disc today. 18 (19), 1007-1013 (2013).</li><li>Wen, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Applications+of+differential+scanning+calorimetry+for+thermal+stability+analysis+of+proteins:+qualification+of+DSC.">Applications of differential scanning calorimetry for thermal stability analysis of proteins: qualification of DSC.</a> J. Pharm. Sci. 101 (3), 955-964 (2012).</li></ol>

所有的作家都在赛诺菲巴斯德的员工。这项工作是由赛诺菲巴斯德公司资助，并为这个视频文章出版费用由赛诺菲巴斯德公司支付。作者有没有相关的隶属关系或与拥有的手稿中讨论的主题或材料的财务冲突的任何组织或实体资金介入。这些措施包括就业，顾问，股权或期权，或特许权使用费。 没有写作援助用于生产该手稿。

这个过程已经在很多特性测试包，包括稳定和可比性的产品研究21被成功纳入。在实时稳定性研究，DSC用于监视Tm 值 ，以及随时间估计生物制剂的F- 一个 ，以确定其保质期。至于产品可比性，它是用来评估过程和设施变化带来的影响，以及对大量生产的结构构象关键制造步骤的效果。这通常涉及产生大量的ΔH，以已被指定为理想产品的参考产品的直接比较。此外，DSC已被证明是对产品配方研究37的有用的分析工具。在不同的缓冲液和不同浓度的蛋白质的Tm 值可用于确定的醒最稳定到制剂的蛋白质。 为了确保这一方法和结果的客观性的可靠性，以保持测试的参数保持一致，从运行到相同的研究（ 如疫苗配方研究）中运行是非常重要的。然而，程序可以被修改，以适应各种蛋白质的物理性质的差异。可以作出变形的一个例子是改变该实验38，39的扫描速率。该均倾向于形成聚集体加热时以更快的扫描速度进行了检查蛋白质（ 例如，120℃/小时），以避免聚集物的热过渡轮廓的贡献以及堵塞量热计的毛细管。值得一提的是，扫描速率可以影响的DSC实验38的结果。热转变峰的加宽已经观察到，在一些亲增加扫描速率teins;然而，Tm 值相当稳定38。此外，样品制备透析和脱气步骤也是准确的结果31非常关键。透析可以确保在样品和缓冲液的组合物中的唯一的区别是蛋白;因此，所有被样品吸收的多余的热量可以归因于蛋白质的热容量。脱气确保精确体积的分析，作为热力学参数的推断假定展开事件下恒定体积和压力31发生。假设的恒定压部是由系统的氮气加压占按程序的第1.1节。 相比于测定蛋白质的构象，如圆二色性（CD）和荧光谱研究的稳定性的其他的方法，DSC在一个COM提供了许多的优点商用环境，包括节省成本和时间。首先，差示扫描热量的绝热设计允许具有更好的温度精度的热稳定性的测定相比，与仪器用于CD和荧光谱研究6测量。其次，与光盘，DSC数据的精度不依赖于蛋白39，40的螺旋;然而，CD提供了有关的二级结构，这将是互补的DSC 41的展开的附加信息。此外，DSC系统的加压使得具有宽的温度范围内未经煮沸样品测试;因此，广泛的蛋白质可以通过DSC进行测试。 而DSC是一种相对快速和简单的方法来确定生物制剂的热稳定性，但也不是没有局限性。首先，基行减步骤介绍了一些形式的人类不一致性到的原始数据分析;因此，在结果中的变化可能不同用户之间被观察到。第二，差示扫描量热仪有可能难以实现在批量制造规模最小浓度限制。第三，不可逆热变性的ΔH并不是绝对的;这意味着，在类似的情况下衍生ΔG（蛋白质稳定性的指标）可能会产生误导。此外，该方法最适合纯化的样品。杂质存在既可以导致在T 微米的移位是否存在与所研究的蛋白质，或新的热转变的外观的相互作用，如果没有相互作用。在任何情况下对这些温谱图额外功能可以被错误地归结为样本，从而影响结果的解释。尽管有这些限制，DSC仍然是一个可靠的方法，它可以提供有关详细个热力学信息E蛋白，如果实施得当42展开的过程。 总之，DSC提供了相当大的优势，作为疫苗产品及其中间体的构象读取工具。的两个参数，以及T M与ΔH，收集大量的同一产品的阵列可以变得可用于检查过程中的变化，制剂和在三级结构的储存条件和蛋白质的稳定性的影响进行了实证基线和病毒抗原21，43。

差扫描量热法（DSC）是直接测量调节的温度变化1，2，3，4，5，6，7，8，9中发生在相对于参照样品中热能的吸收的差的实验方法，10，11，12。在差示扫描量热计进行的，该方法包括引入热能到样品单元和参考单元同时而相同地同时增加电池的温度随时间2， 第13，14。由于在样品和参考的组合物差，不同量的能量将需要提高电池2，12，13的温度。因此，为了补偿电池之间的温度差所需要的能量的过剩量被测量并直接与样品1,3的具体热力学性质。 在20世纪60年代，MJ O&#39;Neil的和珀金埃尔默的大肠杆菌沃森开发的第一个差示扫描量热计来测量固体材料2，3，4的热流。与此同时，PL普里瓦洛夫与物理研究所博士Monaseldze EL格鲁吉亚共和国（前苏联）创建了一个可以FO可以使用的唯一差别绝热量热- [R生化研究5,6。随后，Andronikashvili球队在物理研究所，格鲁吉亚共和国，报道生物分子，如纤维和球状蛋白质，DNA，和RNA使用DSC 7，8，9的热容量。通过斯特体范特10，11，12，Brandts 13和普里瓦洛夫14，15，16，使一些队集中在理论和DSC的实际应用的发展，以调查的蛋白解折叠热力学细节。 DSC的学习大的超分子结构，例如噬菌体，叶绿体，磷脂液晶，和肉蛋白的值也有报道17 </<html> SUP&gt;，18，19，20。 现在的DSC已成为药物研究和发展的普遍用于生物分子的热稳定性的评价，特别是蛋白质1，21，22。这主要是由于进步在用于进行实验23中 ，24中的仪器的灵敏度和自动化方面。这里，在DSC实验，即，摩尔热容作为温度的函数的最终结果，用于估计下面热力学参数（在热容（ΔCp），焓（ΔH），熵变（ΔS）和吉布斯自由能（ΔG））使用以下方程: eq1.jpg"/&gt;（1） <img alt="公式（2）" src="/files/ftp_upload/55262/55262eq2.jpg" /> （2） <img alt="公式3" src="/files/ftp_upload/55262/55262eq3.jpg" /> （3） <img alt="公式4" src="/files/ftp_upload/55262/55262eq4.jpg" /> （4） <img alt="公式5" src="/files/ftp_upload/55262/55262eq5.jpg" /> （5） 其中Cp测量热容量; q为热流进入测试材料; T 0的和T分别是22，25的过渡的初始和最终温度。还值得一提的是，上述公式适用于可进行两种状态过渡和可逆热去折叠22单域蛋白质。更复杂的蛋白质（ 例如 ，非两态蛋白质和低聚物）的分析-以HAVE由Friere 等人的报道。 26;约翰逊等人。 27;和Kasimova 等 。 28。 为了确定蛋白质是否经历两个状态转换或形成热变性过程中的中间体，该实验得出的焓（ΔH;也称作量热焓ΔHCAL）进行比较，以使用下面给出（也是范特霍夫等式导出的焓称为范特霍夫焓;ΔHV H）: <img alt="公式6" src="/files/ftp_upload/55262/55262eq6.jpg" /> （6） 其中，T m是过渡的中点温度，R是理想气体常数（1.987卡摩尔-1 K -1）和Y是在展开状态16中 ，29中的蛋白群的分数。如果ΔHVH为等于ΔH 校准 ;或ΔHVH /ΔH 卡尔等于1，则蛋白质经历了一个"全或无"的过渡（ 即两个国家的过渡）16，25，29。然而，如果ΔHVH小于ΔH 校准 ;或ΔHVH /ΔH 校准小于1，所述蛋白质经历非二状态转变16，25，29。 ΔHVH /ΔH 校准的比例也相应于熔化作为热力学协作单位或结构域26的蛋白质结构的比例。 以上这样提到ΔG和ΔH的热力学参数提供有关蛋白质的热稳定性，包括生物制剂的有用信息<html> 30。然而，重点是对T M和ΔH本出版物中规定，因为他们是这个协议的报告值。 Tm 值是过渡，其中，折叠和蛋白质的未折叠状态在平衡状态（ 即 ，ΔG= 0）25，31的中点温度。该蛋白质的Tm 值越高，其热稳定性31越高。 ΔH对应于热容量对温度曲线（也称为热分析）的峰值（多个）下的面积在DSC实验16，25的端部产生。它是使蛋白质变性，并且可以用来估计在蛋白质制剂中的活性级分（F a）需要的能量（ 即，与样品中活性构象的蛋白的比例），使用以下等式: jove_content"&gt; <img alt="公式7" src="/files/ftp_upload/55262/55262eq7.jpg" /> （7） 其中，ΔH是蛋白质样品的实验得出的焓和Q为良好表征的参考或标准化蛋白22所确定的热焓。 F A的估计是根据需要由ICH指南32胁迫条件下监测产品的实时稳定性以及进行稳定性研究显著。 ΔH的比较也提供了有关的蛋白31的三级结构构的紧凑性的信息。 该协议细节的程序评估在工业环境中的蛋白质的热稳定性，并已被广泛用于疫苗制剂。它是使用产生可重复的结果℉的自动化差扫描热量计开发或蛋白质浓度低至300微克/毫升。

<table><tbody><tr><td>Differential Scanning Calorimeter</td><td>Malvern Instruments Ltd</td><td>28428948 (Via GE Healthcare)</td><td>Has an autosampler for automated dispensing of samples into the cell to reduce human effort and errors.</td></tr><tr><td>Contrad 100</td><td>Decon Laboratories Inc</td><td>1504</td><td>Dilute with water to 20% before use</td></tr><tr><td>500 &amp;micro;L Polypropylene round bottom 96 well plate</td><td>Canadian Life Science</td><td>ML072100</td><td>Equivalent plates from other suppliers (&lt;em&gt;e.g.&lt;/em&gt;, VW) can also be used</td></tr><tr><td>MicroCal ThermoVac&amp;nbsp;</td><td>Malvern Instruments Ltd</td><td>N/Ap</td><td>provided with the Cap VP DSC</td></tr><tr><td>Biosafety cabinet&amp;nbsp;</td><td>Labconco</td><td>Logic+ - A2</td><td>biocontainment laminar flow cabinet for sample preparation</td></tr><tr><td>Slide-A-Lyzer dialysis cassette</td><td>Thermo Scientific</td><td>66810 or 66380</td><td>to equilibrate the sample and buffer</td></tr></tbody></table>

differential scanning calorimetry &#8212; a method for assessing the thermal stability and conformation of protein antigen

差扫描量热法（DSC）是一种分析技术，其测量样品的摩尔热容与温度的函数关系。在蛋白质样品的情况下，DSC型材提供关于热稳定性的信息，并在一定程度上可作为可用于评估结构构象的结构的"指纹"。 （; Tm 值熔融温度）和破坏的相互作用稳定三级结构所需的能量;蛋白质（焓ΔH）它使用其测量热转变温度差示扫描量热计进行。比较是制剂以及生产批次之间进行，并在得到的值的差异表示在热稳定性和结构构象差异。数据说明在工业环境中的稳定性研究使用DSC，以及监控关键制造步骤提供，因为这亲的有效性的证明母育。相较于其他的方法用于评估蛋白质构象的热稳定性，DSC是具有成本效益的，需要很少的样品制备步骤，并且还提供了蛋白质解折叠过程的一个完整的热力学轮廓。

从最DSC实验的原始数据表示为一个热通量与温度图，作为量热计实际测量热流到样品溶液的速率的差异和缓冲器35。因此，如果两个小区（ 即样品和参考细胞）的实验期间包含相同的解决方案，从扫描的原始数据应与没有观察到峰的扁平线。观察到的任何峰可以归因于仪器错误（ 例如 ，损坏或污染的细胞），这是为什么运行缓冲扫描样品之前分析是足够的系统适用性试验。 图1示出指示该量热计在样品分析前良好的工作状态的典型缓冲扫描的结果。 图2显示了差异为的DSC实验的原始数据进行erent很多两条蛋白样品。如前面暗示，观察到的峰是在样品和它们各自的缓冲区的热通量的差异。在样品浓度的差异可引起由热量计记录的热容量的变动;但是，这些变化是样品分析按程序的第4.2节中归一化。更高的浓度也可以显示其他的热力学结构域在较低浓度没有贡献的过渡。此外，每个过渡表示热力学域可包括蛋白质36的一个或多个结构域。在这种情况下，蛋白1具有熔融协同三个结构域。 图3示出了从蛋白1和2在图2中， 即呈现的原始数据的分析所产生的结果，基线减法和迭代曲线音响后拟合。所得热分析已归一扫描速率和浓度（自动在分析软件预先设定的算法进行）;因此，呈现的实验的结果中可比较的热容量对温度的曲线图。分析软件从热容量对温度的图，如T 微米 ΔCp使用数据，以得到使用取决于蛋白质的折叠的协同上面给出的方程的变化等热力学参数。 当测试未知样品，设置适当的温度范围内是至关重要的。否则，不完整的热分析可能导致，如在图4中示出。虽然由这种配置的Tm可以得出，ΔH不能准确地确定。因此，样品必须具有较大的温度范围内进行复检完全捕获热转变。一些蛋白质也重新完全变性后adily形式聚集，导致增加过渡后的热容量: 如图2B所示的本常表现为不完整的热分析图。然而，具有较高的最终温度复检可以帮助确认是否有在热分析的该区域的构象转变的发生或它仅仅是蛋白质聚集体的热吸收效果。 热稳定性是在行业37的蛋白质和蛋白质基的产品的最显著物理性能之一。在医药品，它是用来确定不同的条件，包括配制剂缓冲液和环境因素，如湿度和温度下生物制剂的稳定性。它也可以用来监控关键制造步骤（ 例如，纯化和解毒），以确保生产批次之间的构象的一致性。 <strong<html> &gt;图5和6示出了使用的DSC分别以审查的稳定性和两种不同的蛋白质的结构构象的化学解毒和储存条件效果。在Tm和ΔH的显著差异分别表示的构象变化和蛋白质降解。另外，在图6中的第三过渡的损失进一步示出了通过在分子量降低证实当样品用大小排阻层析用多角度光散射（SEC-MALS）进行分析的域的降解（数据未示出）。 <img alt="图1" src="/files/ftp_upload/55262/55262fig1.jpg" /> 图1:缓冲扫描。在没有观察到峰每次扫描的梯度相似性表明仪器处于良好的工作状态，并产生重复的结果。//ecsource.jove.com/files/ftp_upload/55262/55262fig1large.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。 <img alt="图2" src="/files/ftp_upload/55262/55262fig2.jpg" /> 图2:原始数据从DSC实验收集。这些图是实验运行后获得的未分析（原始）数据（ 即前基线扣除和曲线拟合）良好的表现。每一行代表一个生产批次。蛋白2趋于在加热时更容易地聚集，从而在热分析的后过渡区域的增加在100℃以上的热容量。 <a href="http://ecsource.jove.com/files/ftp_upload/55262/55262fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/55262/55262fig3.jpg" /><br/&gt; 图3:DSC分析数据。这些图表进行分析DSC数据的良好表现（基线扣除和曲线拟合后， 即 ）。蓝线代表基线减法之后的温谱图，而红线代表最佳配合到温谱图的曲线。 （一）蛋白质1样品＃12 的Tm和ΔH是80.16°C和1.69×10 6千卡/摩尔分别。 （B）的T M和ΔH蛋白质1样品＃13是80.15°C和1.71×10 6千卡/摩尔分别。 （C）蛋白质2样品＃21 的Tm和ΔH是75.01°C和4.08×10 6千卡/摩尔分别。 （D）Tm 值和ΔH蛋白质2样品＃22 75.67°C和4.22×10 6千卡/摩尔分别。 <a href="http://ecsource.jove.com/files/ftp_upload/55262/55262fig3large.jpg" target="_blank">请点击此处</a>查看该图的放大版本。 <img alt="图4" src="/files/ftp_upload/55262/55262fig4.jpg" /> 图4:不完整的热像图。原始数据收集在一个温度范围内充分分析蛋白1。实验的最终温度设定为90℃相比，实验用于图2A将其设定为120℃，这并没有容纳该蛋白质的整个过渡轮廓。 <a href="http://ecsource.jove.com/files/ftp_upload/55262/55262fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/55262/55262fig5.jpg" /> 图5:分析数据显示化工解毒之功效蛋白质1（A）蛋白1 的三级结构是其天然构象和毒素公顷■在56.84℃，它的T M和ΔH在2.57×10 5千卡/摩尔。 （B）蛋白1（ 即类毒素）的解毒形式分别是T M和81.01的℃，ΔH值和1.89×10 6千卡/摩尔。因此，可以得出结论，在解毒步骤中引入某种形式的变化，以蛋白1的结构构象，其赋予更大的稳定性（较高的Tm）至其去毒的形式。 <a href="http://ecsource.jove.com/files/ftp_upload/55262/55262fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图6" src="/files/ftp_upload/55262/55262fig6.jpg" /> 图6:分析的数据显示储存条件的影响3.蛋白的构象这些图表说明储存温度的影响（2 - 8°C）的稳定性和专业三级结构3 TEIN超过30周。 Tm 值和在第 8（A）的蛋白3和存储的38 个星期（B）的 ΔH值在下面的表1中给出。 <a href="http://ecsource.jove.com/files/ftp_upload/55262/55262fig6large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <table fo:keep-together.within-page="1"><tbody><tr><td> 样品 </td><td> 的Tm 1（℃） </td><td> ΔH1（卡/摩尔） </td><td> 的Tm 2（℃） </td><td> ΔH2（卡/摩尔） </td><td> Tm为3（℃） </td><td> ΔH3（卡/摩尔） </td></tr><tr><td>蛋白3保存在2-8°C，持续8周</td><td> 61.91 </td><td> 1.71×10 5个 </td><td> 75.54 </td><td> 2.17×10 5个 </td><td> 90.29 </td><td> 4.17×10 5个 </td></tr><tr><tD&gt;蛋白3保存在2-8℃下38周</td><td> 61.87 </td><td> 1.66×10 5个 </td><td> 75.18 </td><td> 1.46×10 5个 </td><td> 90.22 </td><td> 5.76×10 5个 </td></tr></tbody></table> 表1:Tm和ΔH值蛋白3在凌晨2存储8 日和38 日周- 8°C。虽然在这两个时间点的的Tm值是相似的，在ΔH值的差异表明，蛋白质3的三级结构已指定的储存条件下劣化超过30周。

Watch this Scientific Journal Video about Differential Scanning Calorimetry â€” A Method for Assessing the Thermal Stability and Conformation of Protein Antigen at JoVE.com

Differential Scanning Calorimetry &#8212; A Method for Assessing the Thermal Stability and Conformation of Protein Antigen

差示扫描量热法测量，以变性的蛋白质所需要的热转变温度（S）和总的热能量。得到的结果用于评估疫苗制剂的蛋白质抗原的热稳定性。

差示扫描量热 - 用于评估蛋白质抗原的热稳定性和构象的方法

Differential scanning calorimetry (DSC) is an analytical technique that measures the molar heat capacity of samples as a function of temperature. In the ...

differential-scanning-calorimetry-method-for-assessing-thermal

Universidad de Cartagena UDEC

Research

Education

JoVE Core

JoVE Journal

Physics

Biochemistry

Unit 1

The First Law of Thermodynamics

SCIENTISTS IN ACTION

38.6K 次观看.  Sanofi Pasteur Limited. 差示扫描量热法测量，以变性的蛋白质所需要的热转变温度（S）和总的热能量。得到的结果用于评估疫苗制剂的蛋白质抗原的热稳定性。 

视频: Differential Scanning Calorimetry — A Method for Assessing the Thermal Stability and Conformation of Protein Antigen

Measuring Biomolecular DSC Profiles with Thermolabile Ligands to Rapidly Characterize Folding and Binding Interactions

Differential scanning calorimetry (DSC) is a powerful technique for quantifying thermodynamic parameters governing biomolecular folding and binding interactions. This information is critical in the design of new pharmaceutical compounds. However, many pharmaceutically relevant ligands are chemically unstable at the high temperatures used in DSC analyses. Thus, measuring binding interactions is challenging because the concentrations of ligands and thermally-converted products are constantly changing within the calorimeter cell. Here, we present a protocol using thermolabile ligands and DSC for rapidly obtaining thermodynamic and kinetic information on the folding, binding, and ligand conversion processes. We have applied our method to the DNA aptamer MN4 that binds to the thermolabile ligand cocaine. Using a new global fitting analysis that accounts for thermolabile ligand conversion, the complete set of folding and binding parameters are obtained from a pair of DSC experiments. In addition, we show that the rate constant for thermolabile ligand conversion may be obtained with only one supplementary DSC dataset. The guidelines for identifying and analyzing data from several more complicated scenarios are presented, including irreversible aggregation of the biomolecule, slow folding, slow binding, and rapid depletion of the thermolabile ligand.

We present a protocol for rapid characterization of biomolecular folding and binding interactions with thermolabile ligands using differential scanning calorimetry.

用耐热配体测量生物分子 DSC 剖面快速表征折叠和结合的相互作用

Differential scanning calorimetry (DSC) is a powerful technique for quantifying thermodynamic parameters governing biomolecular folding and binding ...

科研

生物化学

Designing Silk-silk Protein Alloy Materials for Biomedical Applications

Fibrous proteins display different sequences and structures that have been used for various applications in biomedical fields such as biosensors, nanomedicine, tissue regeneration, and drug delivery. Designing materials based on the molecular-scale interactions between these proteins will help generate new multifunctional protein alloy biomaterials with tunable properties. Such alloy material systems also provide advantages in comparison to traditional synthetic polymers due to the materials biodegradability, biocompatibility, and tenability in the body. This article used the protein blends of wild tussah silk (Antheraea pernyi) and domestic mulberry silk (Bombyx mori) as an example to provide useful protocols regarding these topics, including how to predict protein-protein interactions by computational methods, how to produce protein alloy solutions, how to verify alloy systems by thermal analysis, and how to fabricate variable alloy materials including optical materials with diffraction gratings, electric materials with circuits coatings, and pharmaceutical materials for drug release and delivery. These methods can provide important information for designing the next generation multifunctional biomaterials based on different protein alloys.

Blending is an efficient approach to generate biomaterials with a broad range of properties and combined features. By predicting the molecular interactions between different natural silk proteins, new silk-silk protein alloy platforms with tunable mechanical resiliency, electrical response, optical transparency, chemical processability, biodegradability, or thermal stability can be designed.

设计丝绸蚕丝蛋白合金生物医用材料

Fibrous proteins display different sequences and structures that have been used for various applications in biomedical fields such as biosensors, ...

生物工程

How to Stabilize Protein: Stability Screens for Thermal Shift Assays and Nano Differential Scanning Fluorimetry in the Virus-X Project

The Horizon2020 Virus-X project was established in 2015 to explore the virosphere of selected extreme biotopes and discover novel viral proteins. To evaluate the potential biotechnical value of these proteins, the analysis of protein structures and functions is a central challenge in this program. The stability of protein sample is essential to provide meaningful assay results and increase the crystallizability of the targets. The thermal shift assay (TSA), a fluorescence-based technique, is established as a popular method for optimizing the conditions for protein stability in high-throughput. In TSAs, the employed fluorophores are extrinsic, environmentally-sensitive dyes. An alternative, similar technique is nano differential scanning fluorimetry (nanoDSF), which relies on protein native fluorescence. We present here a novel osmolyte screen, a 96-condition screen of organic additives designed to guide crystallization trials through preliminary TSA experiments. Together with previously-developed pH and salt screens, the set of three screens provides a comprehensive analysis of protein stability in a wide range of buffer systems and additives. The utility of the screens is demonstrated in the TSA and nanoDSF analysis of lysozyme and Protein X, a target protein of the Virus-X project.

A protocol is presented to rapidly test the thermal stability of proteins in a variety of conditions through thermal shift assays and nano differential scanning fluorimetry. Buffer systems, salts and additives, together comprising three unique stability screens, are assayed with proteins to identify suitable buffers for functional and structural studies.

如何稳定蛋白质: 病毒 x 项目中热移检测和纳米差动扫描荧光测量的稳定性筛选

The Horizon2020 Virus-X project was established in 2015 to explore the virosphere of selected extreme biotopes and discover novel viral proteins. To ...

Nano-Differential Scanning Fluorimetry for Screening in Fragment-based Lead Discovery

Thermal shift assays (TSAs) examine how the melting temperature (Tm) of a target protein changes in response to changes in its environment (e.g., buffer composition). The utility of TSA, and specifically of nano-Differential Scanning Fluorimetry (nano-DSF), has been established over the years, both for finding conditions that help stabilize a specific protein and for looking at ligand binding by monitoring changes in the apparent Tm. This paper presents an efficient screening of the Diamond-SGC-iNEXT Poised (DSi-Poised) fragment library (768 compounds) by the use of nano-DSF, monitoring Tm to identify potential fragment binding. The prerequisites regarding protein quality and concentration for performing nano-DSF experiments are briefly outlined followed by a step-by-step protocol that uses a nano-liter robotic dispenser commonly used in structural biology laboratories for preparing the required samples in 96-well plates. The protocol describes how the reagent mixtures are transferred to the capillaries needed for nano-DSF measurements. In addition, this paper provides protocols to measure thermal denaturation (monitoring intrinsic tryptophan fluorescence) and aggregation (monitoring light back-scattering) and the subsequent steps for data transfer and analysis. Finally, screening experiments with three different protein targets are discussed to illustrate the use of this procedure in the context of lead discovery campaigns. The overall principle of the method described can be easily transferred to other fragment libraries or adapted to other instruments.

Monitoring changes in the melting temperature of a target protein (i.e., thermal shift assay, TSA) is an efficient method for screening fragment libraries of a few hundred compounds. We present a TSA protocol implementing robotics-assisted nano-Differential Scanning Fluorimetry (nano-DSF) for monitoring intrinsic tryptophan fluorescence and light back-scattering for fragment screening.

纳米微分扫描荧光仪，用于在基于碎片的铅发现中筛选

Thermal shift assays (TSAs) examine how the melting temperature (Tm) of a target protein changes in response to changes in its environment (e.g., buffer ...

A Package of Established Analytical Tools to Investigate the Solid-State Alteration of Lipid-Based Excipients

Lipid-based excipients (LBEs) are low-toxic, biocompatible, and natural-based, and their application supports the sustainability of pharmaceutical manufacturing. However, the major challenge is their unstable solid-state, affecting the stability of the pharmaceutical product. Critical physical properties of lipids for their processing-such as melt temperature and viscosity, rheology, etc.-are related to their molecular structure and their crystallinity. Additives, as well as thermal and mechanical stress involved in the manufacturing process, affect the solid-state of lipids and thus the performance of pharmaceutical products thereof. Therefore, understanding the alteration in the solid-state is crucial. In this work, the combination of powder x-ray diffraction and differential scanning calorimetry (DSC) is introduced as the gold standard for the characterization of lipids' solid state. X-ray diffraction is the most efficient method to screen polymorphism and crystal growth. The polymorphic arrangement and the lamella length are characterized in the wide- and small-angle regions of x-ray diffraction, respectively. The small-angle x-ray scattering (SAXS) region can be further used to investigate crystal growth. Phase transition and separation can be indicated. DSC is used to screen the thermal behavior of lipids, estimate the miscibility of additives and/or active pharmaceutical ingredients (API) in the lipid matrix, and provide phase diagrams. Four case studies are presented in which LBEs are either used as a coating material or as an encapsulation matrix to provide lipid-coated multiparticulate systems and lipid nanosuspensions, respectively. The lipid solid-state and its potential alteration during storage are investigated and correlated to the alteration in the API release. Qualitative microscopical methods such as polarized light microscopy and scanning electron microscopy are complementary tools to investigate micro-level crystallization. Further analytical methods should be added based on the selected manufacturing process. The structure-function-processability relationship should be understood carefully to design robust and stable lipid-based pharmaceutical products.

This publication shows the application of x-ray diffraction and differential scanning calorimetry as gold standards for investigating the solid-state of lipid-based excipients (LBEs). Understanding the solid-state alteration in LBEs and its effect on the performance of pharmaceutical products thereof is the key factor for manufacturing robust lipid-based dosage forms.

一套成熟的分析工具，用于研究脂质赋形剂的固态变化

Lipid-based excipients (LBEs) are low-toxic, biocompatible, and natural-based, and their application supports the sustainability of pharmaceutical ...

化学

<meta charset="utf8"></head><body>用于蛋白质-配体相互作用的高通量热位移检测

Utilizing Thermal Shift Assay to Probe Substrate Binding to Selenoprotein O

Defining the biological importance of proteins with unknown functions poses a significant obstacle in understanding cellular processes. Although bioinformatic and structural predictions have contributed to the study of unknown proteins, in vitro experimental validations are often hampered by the optimal conditions and cofactors required for biochemical activity. Cofactor binding is not only essential for the activity of some enzymes but may also enhance the thermal stability of the protein. One practical application of this phenomenon lies in utilizing the change in thermal stability, as measured by alterations in the protein's melting temperature, to probe ligand binding. 
Thermal shift assay (TSA) can be used to analyze the binding of different ligands to the protein of interest or find a stabilizing condition to perform experiments such as X-ray crystallography. Here we will describe a protocol for TSA utilizing the pseudokinase, Selenoprotein O (SelO), for a simple and high-throughput method for testing metal and nucleotide binding. In contrast to canonical kinases, SelO binds ATP in an inverted orientation to catalyze the transfer of AMP to the hydroxyl side chains of proteins in a posttranslational modification known as protein AMPylation. By leveraging the shift in the melting temperatures, we provide crucial insights into the molecular interactions underlying SelO function.

<meta charset="utf8"></head><body>作者聚焦：通过热位移分析推进蛋白质的结构和生化研究

Here, we present the thermal shift assay, a high-throughput, fluorescence-based technique used to investigate the binding of small molecules to proteins of interest.

利用 Thermal Shift 分析探测底物与 Selenoprotein O 的结合

Defining the biological importance of proteins with unknown functions poses a significant obstacle in understanding cellular processes. Although ...

Determination of Protein-ligand Interactions Using Differential Scanning Fluorimetry

A wide range of methods are currently available for determining the dissociation constant between a protein and interacting small molecules. However, most of these require access to specialist equipment, and often require a degree of expertise to effectively establish reliable experiments and analyze data. Differential scanning fluorimetry (DSF) is being increasingly used as a robust method for initial screening of proteins for interacting small molecules, either for identifying physiological partners or for hit discovery. This technique has the advantage that it requires only a PCR machine suitable for quantitative PCR, and so suitable instrumentation is available in most institutions; an excellent range of protocols are already available; and there are strong precedents in the literature for multiple uses of the method. Past work has proposed several means of calculating dissociation constants from DSF data, but these are mathematically demanding. Here, we demonstrate a method for estimating dissociation constants from a moderate amount of DSF experimental data. These data can typically be collected and analyzed within a single day. We demonstrate how different models can be used to fit data collected from simple binding events, and where cooperative binding or independent binding sites are present. Finally, we present an example of data analysis in a case where standard models do not apply. These methods are illustrated with data collected on commercially available control proteins, and two proteins from our research program. Overall, our method provides a straightforward way for researchers to rapidly gain further insight into protein-ligand interactions using DSF.

Differential scanning fluorimetry is a widely used method for screening libraries of small molecules for interactions with proteins. Here, we present a straightforward method to extend these analyses to provide an estimate of the dissociation constant between a small molecule and its protein partner.

蛋白质 - 配体相互作用采用差示扫描荧​​光法测定

A wide range of methods are currently available for determining the dissociation constant between a protein and interacting small molecules. However, most ...

生物学

使用荧光高灵敏度检测化合物-蛋白质结合

Detection of CD40 Protein-Umbelliferone Interaction via Differential Scanning Fluorescence

The investigation of interactions between different molecules is a crucial aspect of understanding disease pathogenesis and screening for drug targets. Umbelliferone, an active ingredient in Tibetan medicine Vicatia thibetica, exhibits an immunomodulatory effect with an unknown mechanism. The CD40 protein is a key target in the immune response. Therefore, this study employs the principle of differential scanning fluorescence technology to analyze the interactions between CD40 protein and umbelliferone using fluorescent enzyme markers. Initially, the stability of the protein fluorescent orange dye was experimentally verified, and the optimal dilution ratio of 1:500 was determined. Subsequently, it was observed that the temperature melting (Tm) value of CD40 protein tended to decrease with an increase in concentration. Interestingly, the interaction between CD40 protein and umbelliferone was found to enhance the thermal stability of CD40 protein. This study represents the first attempt to detect the binding potential of small molecule compounds and proteins using fluorescence microplates and fluorescent dyes. The technique is characterized by high sensitivity and accuracy, promising advancements in the fields of protein stability, protein structure, and protein-ligand interactions, thus facilitating further research and exploration.

作者聚焦:简化 PCR 方法以提高可及性和效率

This protocol describes a unique method for detecting the binding between compounds and protein molecules, offering the advantages of minimal protein sample loss and high data accuracy.

通过差示扫描荧光检测CD40蛋白-umbellefone相互作用

The investigation of interactions between different molecules is a crucial aspect of understanding disease pathogenesis and screening for drug targets. ...

差示扫描量热 - 用于评估蛋白质抗原的热稳定性和构象的方法

摘要

探索更多视频

此视频中的章节

用耐热配体测量生物分子 DSC 剖面快速表征折叠和结合的相互作用

设计丝绸蚕丝蛋白合金生物医用材料

如何稳定蛋白质: 病毒 x 项目中热移检测和纳米差动扫描荧光测量的稳定性筛选

纳米微分扫描荧光仪，用于在基于碎片的铅发现中筛选

一套成熟的分析工具，用于研究脂质赋形剂的固态变化

利用 Thermal Shift 分析探测底物与 Selenoprotein O 的结合

利用 Thermal Shift 分析探测底物与 Selenoprotein O 的结合

利用 Thermal Shift 分析探测底物与 Selenoprotein O 的结合

蛋白质 - 配体相互作用采用差示扫描荧光法测定

通过差示扫描荧光检测CD40蛋白-umbellefone相互作用

差示扫描量热 - 用于评估蛋白质抗原的热稳定性和构象的方法

摘要

探索更多视频

此视频中的章节

用耐热配体测量生物分子 DSC 剖面快速表征折叠和结合的相互作用

设计丝绸蚕丝蛋白合金生物医用材料

如何稳定蛋白质: 病毒 x 项目中热移检测和纳米差动扫描荧光测量的稳定性筛选

纳米微分扫描荧光仪，用于在基于碎片的铅发现中筛选

一套成熟的分析工具，用于研究脂质赋形剂的固态变化

利用 Thermal Shift 分析探测底物与 Selenoprotein O 的结合

利用 Thermal Shift 分析探测底物与 Selenoprotein O 的结合

利用 Thermal Shift 分析探测底物与 Selenoprotein O 的结合

蛋白质 - 配体相互作用采用差示扫描荧​​光法测定

通过差示扫描荧光检测CD40蛋白-umbellefone相互作用

蛋白质 - 配体相互作用采用差示扫描荧光法测定