Western Blotting for Staphylococcus aureus AgrA
葡萄球菌属AgrA蛋白印迹实验
发布: 2014年03月20日第4卷第6期 DOI: 10.21769/BioProtoc.1071 浏览次数: 13714
评审: Fanglian HeAnonymous reviewer(s)
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参见作者原研究论文
The authors used this protocol in:
Apr 2012
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Abstract
Staphylococcus aureus has a quorum sensing system to regulate the expression of various virulence factors, which is exerted by the agr locus that encodes agrBDCA and a regulatory RNA called RNAIII. AgrB, AgrD, and AgrC proteins are involved in producing and recognizing extracellular quorum sensing molecules and transduce the signal by altering the phosphorylation status of AgrA, which is a positive transcription factor, to regulate cytolysin genes as well as the RNAIII gene. RNAIII regulates the expression of various virulence genes. Expression of the agr locus has been examined in depth at the transcriptional level, but investigations of translational expression are limited, because immunoglobulin G used to detect a specific protein highly reacts to S. aureus protein A. Here, we report a method to detect AgrA that is the transcription factor encoded by the agr regulatory system. Although this is a specific protocol for western blotting of S. aureus AgrA protein, it can also be used for other S. aureus proteins by using the appropriate antibody.
Keywords: AgrA (阿格拉)
Immunoglobulin G (免疫球蛋白G)
Protein A (蛋白)
Western blot analysis (Western blot分析)
Materials and Reagents
- agr-positive S. aureus strains
- BactoTM Tryptic Soy Broth (BD, catalog number: 211825 )
- Lysostaphin (Wako Chemicals USA, catalog number: 120-04313 )
- 15% sodium dodecyl sulfate (SDS)-polyacrylamide gel
- N-Cyclohexyl-3-aminopropanesulfonic acid (CAPS) (Dojindo Molecular Technologies, catalog number: 343-08321 )
- Methanol
- Immobilon-P (EMD Millipore, catalog number: IPVH304F0 )
- Tris(hydroxymethyl)aminometane (Nacalai Tesque, catalog number: 35434-34 )
- EDTA.2Na (Dojindo Molecular Technologies, catalog number: 345-01865 )
- Hydrochloric acid
- Coomassie Plus Protein Assay Reagent (Thermo Fisher Scientific, catalog number: 23236 )
- Easy Blocker (GeneTex, catalog number: GTX425858 )
- Anti-AgrA IgG that was made in our laboratory (Kaito et al., 2013)
- Anti-rabbit IgG conjugated with alkaline phosphatase (Promega Corporation, catalog number: S3731 )
- Nitro blue tetrazolium/5-bromo-4-chloro-3’-indolyphosphate (NBT/BCIP) (Roche Diagnostics, catalog number: 1681451 )
- PVDF membrane
- TE buffer (see Recipes)
- Lysis buffer (see Recipes)
- Staining buffer (see Recipes)
- 10x SDS sample buffer (see Recipes)
- 10x TBS (see Recipes)
- TBST (see Recipes)
Equipment
- Sonicator (Branson, model: Sonifier 450) with double stepped microtip (3 mm) (Branson, part number: 101-063-212 )
- Plastic container
- Centrifuge machine
- Electrophoresis apparatus
- Power supply
- Wet/Tank Blotting Systems (Bio-Rad Laboratories)
Software
- Image J (1.45 s, NIH)
Procedure
文章信息
版权信息
© 2014 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:
- Kaito, C. and Sekimizu, K. (2014). Western Blotting for Staphylococcus aureus AgrA. Bio-protocol 4(6): e1071. DOI: 10.21769/BioProtoc.1071.
- Kaito, C., Saito, Y., Ikuo, M., Omae, Y., Mao, H., Nagano, G., Fujiyuki, T., Numata, S., Han, X., Obata, K., Hasegawa, S., Yamaguchi, H., Inokuchi, K., Ito, T., Hiramatsu, K. and Sekimizu, K. (2013). Mobile genetic element SCCmec-encoded psm-mec RNA suppresses translation of agrA and attenuates MRSA virulence. PLoS Pathog 9(4): e1003269.
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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 > 微生物信号传导 > 群体感应
生物化学 > 蛋白质 > 免疫检测 > 免疫印迹法(WB )
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