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Western Blotting for Staphylococcus aureus AgrA
葡萄球菌属AgrA蛋白印迹实验   

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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

  1. agr-positive S. aureus strains
  2. BactoTM Tryptic Soy Broth (BD, catalog number: 211825 )
  3. Lysostaphin (Wako Chemicals USA, catalog number: 120-04313 )
  4. 15% sodium dodecyl sulfate (SDS)-polyacrylamide gel
  5. N-Cyclohexyl-3-aminopropanesulfonic acid (CAPS) (Dojindo Molecular Technologies, catalog number: 343-08321 )
  6. Methanol
  7. Immobilon-P (EMD Millipore, catalog number: IPVH304F0 )
  8. Tris(hydroxymethyl)aminometane (Nacalai Tesque, catalog number: 35434-34 )
  9. EDTA.2Na (Dojindo Molecular Technologies, catalog number: 345-01865 )
  10. Hydrochloric acid
  11. Coomassie Plus Protein Assay Reagent (Thermo Fisher Scientific, catalog number: 23236 )
  12. Easy Blocker (GeneTex, catalog number: GTX425858 )
  13. Anti-AgrA IgG that was made in our laboratory (Kaito et al., 2013)
  14. Anti-rabbit IgG conjugated with alkaline phosphatase (Promega Corporation, catalog number: S3731 )
  15. Nitro blue tetrazolium/5-bromo-4-chloro-3’-indolyphosphate (NBT/BCIP) (Roche Diagnostics, catalog number: 1681451 )
  16. PVDF membrane
  17. TE buffer (see Recipes)
  18. Lysis buffer (see Recipes)
  19. Staining buffer (see Recipes)
  20. 10x SDS sample buffer (see Recipes)
  21. 10x TBS (see Recipes)
  22. TBST (see Recipes)

Equipment

  1. Sonicator (Branson, model: Sonifier 450) with double stepped microtip (3 mm) (Branson, part number: 101-063-212 )
  2. Plastic container
  3. Centrifuge machine
  4. Electrophoresis apparatus
  5. Power supply
  6. Wet/Tank Blotting Systems (Bio-Rad Laboratories)

Software

  1. Image J (1.45 s, NIH)

Procedure

  1. Pick up single colony of S. aureus into 5 ml of fresh tryptic soy broth (TSB) and aerobically culture it for 15 h at 37 °C with shaking at 150 rpm.
  2. Inoculate 50 µl overnight culture of S. aureus into 5 ml (1:100 dilution) of fresh TSB and aerobically culture it for 24 h or 15 h at 37 °C with shaking at 150 rpm. A600 of starting and end will be approximately 0.06-0.1 and 6-10, respectively.
  3. Collect 650 µl S. aureus cell culture by centrifugation at 10,000 x g for 1 min at 4 °C and discard the supernatant. Resuspend the cell pellet in 195 µl of TE buffer by vigorous vortexing. Add 5 µl of lysostaphin (1mg/ml) to the cell resuspension and incubate the resuspension at 37 °C for 30 min without agitation.
  4. Sonicate the sample on ice (Branson Sonifier 450; double stepped microtip; duty cycle, constant; output control, 1; time, 15 sec), and centrifuge it at 10,000 x g for 10 min at 4 °C.
    Note: This step is essential to remove the most of protein A that is associated with cell wall.
  5. Measure the amount of protein in the supernatant by the Bradford method using Coomassie Plus Protein Assay Reagent according to the manufacture’s protocol. Equalize the protein concentration of different samples by adding TE buffer. Mix the protein samples with 3x SDS sample buffer.
  6. Boil protein samples for 5 min and electrophorese the samples in 15% sodium dodecyl sulfate (SDS)-polyacrylamide gel at 100 V for 3 h.
    Note: For details of SDS-polyacrylamide gel electrophoresis, please see Kaito et al. (2013).
  7. Dip the Gel in a buffer (10 mM CAPS, 20% methanol) for 10 min. Treat PVDF membrane with methanol for a few seconds and dip it in a buffer (10 mM CAPS, 20% methanol). Set the gel, membrane, and buffer (10 mM CAPS, 20% methanol) using Wet/Tank Blotting Systems and transfer the proteins from the gel to a PVDF membrane at 150 mA for 3 h at 4 °C.
  8. Treat the membrane with 10 ml blocking buffer (TBST containing 5% Easy Blocker) in a plastic container at room temperature with gentle agitation for 1 h.
    Note: The use of Easy Blocker is essential to decrease the reactivity of IgG against protein A in the samples.
  9. Treat the membrane with 10 ml blocking buffer containing 1:1,000 anti-AgrA IgG in a plastic container at room temperature with gentle agitation for 1 h.
    Note: For using antibodies, the appropriate concentration should be determined by serial titration.
  10. After washing with 20 ml TBST for three times of 5 min agitation, treat the membrane with 10 ml blocking buffer containing 1:2,000 anti-rabbit IgG conjugated with alkaline phosphatase in a plastic container at room temperature with gentle agitation for 1 h.
  11. After washing with 20 ml TBST for three times of 5 min agitation, treat the membrane with a staining buffer without agitation for 5 min.
    Note: For detection of proteins by alkaline phosphatase, please see details in Kaito et al (2013).
  12. After bands appear, dip the membrane in TE buffer supplemented with 10 mM EDTA to stop overstaining and then dry the membrane on paper towel.
  13. Measure the band intensity by densitometry scanning (Image J 1.45 s, NIH).

Recipes

  1. TE buffer
    10 mM Tris-HCl (pH 8.0)
    1 mM EDTA (pH 8.0)
  2. Staining buffer
    100 mM Tris-HCl (pH 9.5)
    100 mM NaCl
    50 mM MgCl2
    2% NBT/BCIP
  3. 3x SDS sample buffer
    1.5 ml 1 M Tris-HCl (pH 6.8)
    3 ml 20% SDS
    3 ml Glycerol
    500 µl 1% bromo phenol blue
    1.8 ml 2-mercaptoethanol
  4. 10x TBS
    24.2 g Tris
    87.6 g NaCl
    1 L milliQ
    Adjust pH to 7.6 by adding hydrochloric acid
  5. TBST
    125 ml 10x TBS
    1,168 ml milliQ
    7.5 ml 20% Tween20

Acknowledgments

This protocol was adapted from the original work (Kaito et al., 2013) to provide the detailed procedures. This work was supported by Grants-in-Aid for Scientific Research (23249009, 24590519). This work was supported in part by the Naito Foundation, the Program for Promotion of Fundamental Studies in Health Sciences of the National Institute of Biomedical Innovation (NIBIO), and the Genome Pharmaceutical Institute.

References

  1. Gallagher, S. R. and Emily A. Wiley, E. M. (2008). Current protocols essential laboratory techniques. John Wiley & Sons, Inc.
  2. 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.

简介

金黄色葡萄球菌具有调节各种毒力因子的表达的群体感应系统,其通过编码agrBDCA的agr基因座和调节agrBDCA的基因座发挥作用。 RNA称为RNAIII。 AgrB,AgrD和AgrC蛋白参与产生和识别胞外群体感应分子并通过改变作为阳性转录因子的AgrA的磷酸化状态来转导信号,以调节细胞溶素基因以及RNAIII/em>基因。 调节各种毒力基因的表达。已经在转录水平上深入研究了 agr 基因座的表达,但是翻译表达的研究是有限的,因为用于检测特异性蛋白的免疫球蛋白G对于高度反应。 aureus 蛋白A.在这里,我们报告检测AgrA的方法,所述AgrA是由 agr 调节系统编码的转录因子。虽然这是用于Western印迹的特定方案。 aureus AgrA蛋白,它也可以用于其他的。金黄色葡萄球菌通过使用适当的抗体。

关键字:阿格拉, 免疫球蛋白G, 蛋白, Western blot分析

材料和试剂

  1. agr - 主动 aureus 菌株
  2. Bacto TM 胰蛋白酶大豆肉汤(BD,目录号:211825)
  3. 溶葡萄球菌素(Wako Chemicals USA,目录号:120-04313)
  4. 15%十二烷基硫酸钠(SDS) - 聚丙烯酰胺凝胶
  5. N-环己基-3-氨基丙磺酸(CAPS)(Dojindo Molecular Technologies,目录号:343-08321)
  6. 甲醇
  7. Immobilon-P(EMD Millipore,目录号:IPVH304F0)
  8. 三(羟甲基)氨基甲烷(Nacalai Tesque,目录号:35434-34)
  9. EDTA 2Na(Dojindo Molecular Technologies,目录号:345-01865)
  10. 盐酸
  11. Coomassie Plus Protein Assay Reagent(Thermo Fisher Scientific,目录号:23236)
  12. Easy Blocker(GeneTex,目录号:GTX425858)
  13. 在我们的实验室中制备的抗AgrA IgG(Kaito等人,2013)
  14. 与碱性磷酸酶缀合的抗兔IgG(Promega公司,目录号:S3731)
  15. 硝基蓝四唑/5-溴-4-氯-3'-吲哚磷酸盐(NBT/BCIP)(Roche Diagnostics,目录号:1681451)
  16. PVDF膜
  17. TE缓冲区(参见配方)
  18. 裂解缓冲液(见配方)
  19. 染色缓冲液(见配方)
  20. 10x SDS样品缓冲液(见配方)
  21. 10x TBS(请参阅配方)
  22. TBST(参见配方)

设备

  1. 具有双阶梯微尖(3mm)(Branson,部件号:101-063-212)的超声波仪(Branson,型号:Sonifier 450)
  2. 塑料容器
  3. 离心机
  4. 电泳仪
  5. 电源
  6. 湿/罐印迹系统(Bio-Rad Laboratories)

软件

  1. 图像J(1.45s,NIH)

程序

  1. 拾取单个菌落的 S。 金黄色葡萄球菌加入到5ml新鲜胰蛋白酶大豆肉汤(TSB)中,并在37℃下以150rpm振摇,有氧培养15小时。
  2. 接种50μl过夜培养的S。 金黄色葡萄球菌注射到新鲜TSB的5ml(1:100稀释)中,并在37℃下以150rpm振荡将其有氧培养24小时或15小时。 起始和结束的 600 将分别为约0.06-0.1和6-10。
  3. 收集650μl S.金黄色葡萄球菌细胞培养物中,在4℃下以10,000×g离心1分钟,弃去上清液。通过剧烈涡旋重悬细胞沉淀在195微升TE缓冲液。加入5微升溶葡萄球菌素(1毫克/毫升)到细胞悬浮液,孵育重悬浮在37°C,30分钟,无需搅动。
  4. 在冰上对样品进行超声处理(Branson Sonifier 450;双阶式微尖;占空比,恒定;输出对照,1;时间,15秒),并在4℃下以10,000×g离心10分钟。
    注意:此步骤对于清除与细胞壁相关的大部分蛋白A至关重要。
  5. 使用Coomassie Plus蛋白测定试剂根据制造商的方案,通过Bradford方法测量上清液中的蛋白质的量。通过加入TE缓冲液平衡不同样品的蛋白质浓度。将蛋白质样品与3x SDS样品缓冲液混合
  6. 煮沸蛋白样品5分钟,并将样品在15%十二烷基硫酸钠(SDS) - 聚丙烯酰胺凝胶中在100V下电泳3小时。
    注意:有关SDS聚丙烯酰胺凝胶电泳的详细信息,请参阅Kaito et al。 (2013)。
  7. 将凝胶浸入缓冲液(10mM CAPS,20%甲醇)中10分钟。用甲醇处理PVDF膜几秒钟,并将其浸在缓冲液(10mM CAPS,20%甲醇)中。使用湿/罐印迹系统设置凝胶,膜和缓冲液(10mM CAPS,20%甲醇),并且在4℃下将来自凝胶的蛋白质在150mA下转移至PVDF膜3小时。
  8. 在塑料容器中在室温下用10ml封闭缓冲液(含有5%易阻断剂的TBST)处理膜,温和搅拌1小时。
    注意:使用Easy Blocker对于降低样品中IgG对蛋白A的反应性至关重要。
  9. 在塑料容器中在室温下用缓冲搅拌1小时,用含有1:1,000抗AgrA IgG的10ml封闭缓冲液处理膜。
    注意:对于使用抗体,应通过连续滴定测定合适的浓度。
  10. 用20ml TBST洗涤3次,每次5分钟,搅拌后,在塑料容器中,在室温下,用10ml含有与碱性磷酸酶缀合的抗兔IgG的封闭缓冲液处理膜,温和搅拌1小时。
  11. 用20ml TBST洗涤3次,每次5分钟搅拌后,用染色缓冲液处理膜,不搅拌5分钟。
    注意:为了通过碱性磷酸酶检测蛋白质,请参见Kaito等人(2013)中的细节。
  12. 条带出现后,将膜浸在补充有10mM EDTA的TE缓冲液中以停止过染,然后在纸巾上干燥膜。
  13. 通过光密度测量扫描(Image J 1.45s,NIH)测量条带强度。

食谱

  1. TE缓冲区
    10mM Tris-HCl(pH8.0) 1mM EDTA(pH8.0)
  2. 染色缓冲区
    100mM Tris-HCl(pH9.5)
    100 mM NaCl
    50mM MgCl 2/v/v 2%NBT/BCIP
  3. 3x SDS样品缓冲液
    1.5ml 1M Tris-HCl(pH6.8) 3ml 20%SDS
    3毫升甘油
    500μl1%溴酚蓝
    1.8ml 2-巯基乙醇
  4. 10x TBS
    24.2克Tris
    87.6克NaCl 1 L milliQ
    加入盐酸调节pH至7.6
  5. TBST
    125 ml 10×TBS
    1,168ml milliQ
    7.5ml 20%Tween20

致谢

该协议改编自原始工作(Kaito等人,2013)以提供详细的程序。 这项工作是由科学研究助理赞助(23249009,24590519)。 这项工作部分由奈藤基金会,促进国家生物医学创新研究所(NIBIO)的健康科学基础研究计划和基因组药物研究所支持。

参考文献

  1. Gallagher,S.R。和Emily A.Wiley,E.M。(2008)。 当前协议基本实验室技术。 John Wiley& Sons,Inc.
  2. Kaito,C.,Saito,Y.,Ikuo,M.,Omae,Y.,Mao,H.,Nagano,G.,Fujiyuki,T.,Numata,S.,Han,X.,Obata, Hasegawa,S.,Yamaguchi,H.,Inokuchi,K.,Ito,T.,Hiramatsu,K.and Sekimizu,K。(2013)。 移动遗传元件SCC mec - 编码的 psm-mec RNA抑制 agrA 的翻译并减弱MRSA毒力。 9(4):e1003269。
  • English
  • 中文翻译
免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Kaito, C. and Sekimizu, K. (2014). Western Blotting for Staphylococcus aureus AgrA. Bio-protocol 4(6): e1071. DOI: 10.21769/BioProtoc.1071.
  2. 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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