Isolation of Radiolabeled Poliovirus Particles from H1 HeLa Cells
从H1 HeLa细胞中分离放射性标记骨髓灰质炎病毒颗粒
发布: 2013年08月20日第3卷第16期 DOI: 10.21769/BioProtoc.872 浏览次数: 7858
评审: Fanglian He
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参见作者原研究论文
The authors used this protocol in:
Nov 2012
Abstract
The following protocol describes the isolation of radioactive viral and subviral particles from infected cells. This protocol has been written for isolation of poliovirus particles from H1 HeLa cells. Infection protocol and timing of [35S] methionine labeling and particle collection should be tailored to the virus of interest. Following isolation of the viral particles, the viral proteins present in these particles may be separated by gel electrophoresis and visualized by autoradiography.
Materials and Reagents
- H1 HeLa cells
- Poliovirus
- Sucrose (Life Technologies, InvitrogenTM, catalog number: 15503-022 )
- Methionine free DMEM (Life Technologies, Gibco®, catalog number: 21013 )
- MEM (Life Technologies, Gibco®, catalog number: 11095-080 )
- [35S] Methionine (PerkinElmer, catalog number: NEG772002MC )
- Mineral oil (Sigma-Aldrich, catalog number: M5904 )
- SDS-PAGE gel
- Nonidet P-40 (NP-40) (Sigma-Aldrich, catalog number: N-6507 )
- Phenylmethanesulfonyl fluoride (PMSF) (Sigma-Aldrich, catalog number: P3075-1G )
- 40 U RNAsin® Ribonuclease Inhibitor (Promega Corporation, catalog number: N2111 )
- 4x Laemelli loading buffer
- Tris buffer (see Recipes)
- Lysis buffer (see Recipes)
- Gel Fixative (see Recipes)
- Phosphate Buffered Saline (PBS) (see Recipes)
- PBS+ (see Recipes)
Equipment
- Centrifuge tubes (Beckman Coulter, catalog number: 344059 )
- 1.5 ml Eppendorf tubes
- Peristaltic Pump (Bio-Rad Laboratories, model: 731-9001EDU )
- Ultracentrifuge (Beckman Coulter, model: Optima L-90K )
- SW41 Ti rotor (Beckman Coulter, model: 331362 )
- Fraction collection system (Beckman Coulter, catalog number: 270-331580 )
- Gradient maker (Biocomp Gradient Master, model: 107 )
- Liquid scintillation counter (Beckman Coulter, model: LS6000IC )
- Gel dryer (Bio-Rad Laboratories, model: 583 )
Procedure
文章信息
版权信息
© 2013 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
- Richards, A. and Jackson, W. (2013). Isolation of Radiolabeled Poliovirus Particles from H1 HeLa Cells. Bio-protocol 3(16): e872. DOI: 10.21769/BioProtoc.872.
- Richards, A. L. and Jackson, W. T. (2012). Intracellular vesicle acidification promotes maturation of infectious poliovirus particles. PLoS Pathog 8(11): e1003046.
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分类
微生物学 > 微生物生物化学 > 蛋白质
生物化学 > 蛋白质 > 分离和纯化
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