葡萄球菌属AgrA蛋白印迹实验

Chikara Kaito; Kazuhisa Sekimizu

doi:10.21769/BioProtoc.1071

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Peer-reviewed

Western Blotting for Staphylococcus aureus AgrA

葡萄球菌属AgrA蛋白印迹实验

CK Chikara Kaito email

KS Kazuhisa Sekimizu

发布: 2014年03月20日第4卷第6期 DOI: 10.21769/BioProtoc.1071 浏览次数: 12770

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:

Cover of PLOS Pathogens, featuring study using the protocol.

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
Bacto^TM 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

English

中文翻译

文章信息

版权信息

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.