Fluorometric Estimation of Viral Thermal Stability
荧光法预测病毒热稳定性
发布: 2014年08月05日第4卷第15期 DOI: 10.21769/BioProtoc.1199 浏览次数: 12272
评审: Kanika GeraAnonymous reviewer(s)
下载 PDF 
Q&A
参见作者原研究论文
The authors used this protocol in:
Dec 2013
Abstract
Differential Scanning Fluorimetry (DSF) is a rapid, economical, and a straightforward technique for estimating the thermal stability of proteins. The principle involves the binding of a fluorescent dye to thermally exposed hydrophobic pockets of a protein. The dyes used in this technique are highly fluorescent in a non-polar environment and are quenched when exposed to aqueous solution. The change in fluorescence can be used to follow unfolding of proteins induced by temperature, pH, or chaotropic agents. The method is well characterized for monomeric proteins. Here, we extend the application to supramolecular protein and nucleo-protein complexes using virus particles as an example. SYPRO-orange™ dye is the dye of choice because it is matched for use with q-PCR instruments and the fluorescence response is stable across a wide range of pH and temperatures. Advantages of this technique over standard biophysical methods include the ability for high-throughput screening of biological and technical replicates and the high sensitivity.
Keywords: Biophysics (生物物理学)
Virology (病毒学)
Fluorescence (荧光)
Hydrophobic patches (疏水补丁)
Subunit interactions (亚基的相互作用)
Materials and Reagents
- Purified Virus Like Particles (VLPs) of Adeno-associated virus serotypes 1, 2, 5, 8
- Purified recombinant AAV (rAAV) capsids packaging a green fluorescent protein gene (GFP), rAAV1-GFP, rAAV5-GFP, rAAV8-GFP
- PBS (pH 7.0) at 25 °C
- Citric acid (Thermo Fisher Scientific, catalog number: BP339-500 )
- Disodium phosphate (VWR International, catalog number: BDH4538-1KGP )
- 5,000x SYPRO Orange (Life Technologies, catalog number: S6651 )
- Sodium hydroxide (Thermo Fisher Scientific, catalog number: BP359-500 )
- 12 N hydrochloric acid (EMD Millipore, catalog number: HX0603-3 )
- Sodium chloride (VWR International, catalog number: BDH4534-5KGP )
- Magnesium chloride (Thermo Fisher Scientific, catalog number : BP214-500 )
- Molecular biology grade water
- Citrate-Phosphate buffer (see Recipes)
Equipment
- Rotor Gene-3000, qPCR instrument with 36-well rotor (QIAGEN)
- 0.2 ml PCR tubes (BioExpress, catalog number: T-3013-1FC )
- Aluminum foil
- Pipette tips (10 µl, 200 µl) (VWR International, catalog numbers: 37001-162 and 53508-810 )
- Pipetts (200 µl, 0.1-2.5 µl) (Eppendorf, BioExpress, catalog numbers: P-3015-6 and P-3015-1 )
- pH meter (Accument Basic AB 15 pH meter) (Thermo Fisher Scientific)
- Milli-Q purification system (EMD Millipore)
Software
- Rotor gene software (Version 1.7, Build 94)
- Microsoft Excel 2007 on Windows 7
Procedure
文章信息
版权信息
© 2014 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Rayaprolu, V., Kruse, S., Kant, R., Movahed, N., Brooke, D. and Bothner, B. (2014). Fluorometric Estimation of Viral Thermal Stability. Bio-protocol 4(15): e1199. DOI: 10.21769/BioProtoc.1199.
Download Citation in RIS Format
分类
微生物学 > 微生物生物化学 > 蛋白质
生物化学 > 蛋白质 > 修饰
生物化学 > 蛋白质 > 结构
您对这篇实验方法有问题吗？
在此处发布您的问题，我们将邀请本文作者来回答。同时，我们会将您的问题发布到Bio-protocol Exchange，以便寻求社区成员的帮助。
提问指南
+ 问题描述
写下详细的问题描述，包括所有有助于他人回答您问题的信息（例如实验过程、条件和相关图像等）。
发布问题
