Shigella IpaD and IpaB Surface Localizations
志贺氏杆菌IPaD和IPAB表面定位
发布: 2013年11月20日第3卷第22期 DOI: 10.21769/BioProtoc.975 浏览次数: 11128
评审: Fanglian He
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The authors used this protocol in:
Apr 2013
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Abstract
Shigella uses a type III secretion system to invade host cell and to cause disease. Secretion control and insertion of a translocation pore into cell membrane are critical steps for pathogenesis and are tightly linked to the formation of the needle tip complex formed by the IpaB and IpaD proteins (Veenendaal et al., 2007). Surface localizations of IpaD and IpaB were monitored by FACS analysis according to the localization protocol for Pseudomonas aeruginosa homolog PcrV (Lee et al., 2010).
Keywords: Type 3 secretion system (3型分泌系统)
Tip complex (尖端复杂的)
Shigella (志贺菌)
Flow cytometry (流式细胞仪)
Materials and Reagents
- Shigella strains
- Tryptic Soy Broth (TSB) (VWR International, catalog number: for Europe 1.00525.5000 and for U.S. EM1.00525.5007 )
- Agar (MP Biomedicals, catalog number: 0 210026291 )
- Congo Red (VWR International, catalog number: for Europe 34140.184 )
- Anti-IpaD and -IpaB polyclonal antibodies (house made)
- PBS (Fischer Scientific, catalog number: BP399-20 )
- Paraformaldehyde (PFA) (Sigma-Aldrich, catalog number: P6148 )
- Triton X-100 (VWR International, for Europe catalog number: 1.08603.1000 )
- Trizmabase (Sigma-Aldrich, catalog number: T1503 )
- Bovine serum albumin (BSA) (VWR International, catalog number: 422361V )
- Anti-mouse secondary antibody CF647-conjugated (Sigma-Aldrich, catalog number: SAB4600351 )
- PBS + PFA 4% stock solution (see Recipes)
- Congo Red agar plates (see Recipes)
Equipment
- 250 ml Erlenmeyer
- Microtubes
- CR agar plate
- Polystyrene tube for FACS analysis
- Centrifuge Heating magnetic stirrer
- 37 °C shaker
- Rotator mixer
- Flow cytometry with a four-colour FACS Calibur cytometer (Becton Dickinson and Company)
Procedure
文章信息
版权信息
© 2013 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Schiavolin, L., Meghraoui, A. and Allaoui, A. (2013). Shigella IpaD and IpaB Surface Localizations. Bio-protocol 3(22): e975. DOI: 10.21769/BioProtoc.975.
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分类
微生物学 > 微生物生物化学 > 蛋白质 > 免疫检测
微生物学 > 微生物-宿主相互作用 > 细菌
细胞生物学 > 细胞成像 > 荧光
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