通过热迁移分析进行蛋白质稳定性和配体相互作用的分析。
Analysis of protein stability and ligand interactions by thermal shift assay.
作者信息
Huynh Kathy, Partch Carrie L
机构信息
Department of Chemistry and Biochemistry, University of California Santa Cruz, Santa Cruz, California.
出版信息
Curr Protoc Protein Sci. 2015 Feb 2;79:28.9.1-28.9.14. doi: 10.1002/0471140864.ps2809s79.
Abstract
Purification of recombinant proteins for biochemical assays and structural studies is time-consuming and presents inherent difficulties that depend on the optimization of protein stability. The use of dyes to monitor thermal denaturation of proteins with sensitive fluorescence detection enables rapid and inexpensive determination of protein stability using real-time PCR instruments. By screening a wide range of solution conditions and additives in a 96-well format, the thermal shift assay easily identifies conditions that significantly enhance the stability of recombinant proteins. The same approach can be used as an initial low-cost screen to discover new protein-ligand interactions by capitalizing on increases in protein stability that typically occur upon ligand binding. This unit presents a methodological workflow for small-scale, high-throughput thermal denaturation of recombinant proteins in the presence of SYPRO Orange dye.
摘要
用于生化分析和结构研究的重组蛋白纯化既耗时，又存在一些固有困难，这些困难取决于蛋白稳定性的优化。使用染料通过灵敏的荧光检测来监测蛋白质的热变性，能够利用实时PCR仪器快速且低成本地测定蛋白质稳定性。通过在96孔板中筛选广泛的溶液条件和添加剂，热迁移分析能够轻松识别出可显著提高重组蛋白稳定性的条件。利用配体结合时通常会出现的蛋白质稳定性增加，同样的方法可用作初步的低成本筛选，以发现新的蛋白质-配体相互作用。本单元介绍了在SYPRO Orange染料存在下对重组蛋白进行小规模、高通量热变性分析的方法流程。
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本文引用的文献
1
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Nat Protoc. 2013 Nov;8(11):2309-24. doi: 10.1038/nprot.2013.130. Epub 2013 Oct 24.
2
Differential scanning fluorescence approach using a fluorescent molecular rotor to detect thermostability of proteins in surfactant-containing formulations.使用荧光分子转子的差示扫描荧光法检测表面活性剂配方中蛋白质的热稳定性。
Int J Pharm. 2013 Jan 30;441(1-2):255-60. doi: 10.1016/j.ijpharm.2012.11.035. Epub 2012 Nov 28.
3
Differential scanning calorimetry as a tool for protein folding and stability.差示扫描量热法作为一种研究蛋白质折叠和稳定性的工具。
Arch Biochem Biophys. 2013 Mar;531(1-2):100-9. doi: 10.1016/j.abb.2012.09.008. Epub 2012 Sep 27.
4
A thermal stability assay can help to estimate the crystallization likelihood of biological samples.热稳定性分析有助于评估生物样品的结晶可能性。
Acta Crystallogr D Biol Crystallogr. 2011 Nov;67(Pt 11):915-9. doi: 10.1107/S0907444911036225. Epub 2011 Oct 18.
5
Kinase inhibitor selectivity profiling using differential scanning fluorimetry.使用差示扫描荧光法进行激酶抑制剂选择性分析。
Methods Mol Biol. 2012;795:109-18. doi: 10.1007/978-1-61779-337-0_7.
6
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Methods Enzymol. 2011;493:277-98. doi: 10.1016/B978-0-12-381274-2.00011-X.
7
Identification of a NBD1-binding pharmacological chaperone that corrects the trafficking defect of F508del-CFTR.一种纠正F508del-CFTR转运缺陷的NBD1结合药理学伴侣分子的鉴定。
Chem Biol. 2011 Feb 25;18(2):231-42. doi: 10.1016/j.chembiol.2010.11.016.
8
Use of thermal melt curves to assess the quality of enzyme preparations.利用热融曲线评估酶制剂的质量。
Anal Biochem. 2010 Apr 15;399(2):268-75. doi: 10.1016/j.ab.2009.12.018. Epub 2009 Dec 14.
9
Extrinsic fluorescent dyes as tools for protein characterization.作为蛋白质表征工具的外在荧光染料。
Pharm Res. 2008 Jul;25(7):1487-99. doi: 10.1007/s11095-007-9516-9. Epub 2008 Jan 3.
10
The use of differential scanning fluorimetry to detect ligand interactions that promote protein stability.使用差示扫描荧光法检测促进蛋白质稳定性的配体相互作用。
Nat Protoc. 2007;2(9):2212-21. doi: 10.1038/nprot.2007.321.
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