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In bacteria, interaction of various proteins with DNA is essential for the regulation of specific target gene expression. Electrophoretic mobility shift assay (EMSA) is an in vitro approach allowing for the visualization of these protein-DNA interactions. Rgg proteins comprise a family of transcriptional regulators widespread among the Firmicutes. Some of these proteins function independently to regulate target gene expression, while others have now been demonstrated to function as effectors of cell-to-cell communication, having regulatory activitiesthat that are modulated via direct interaction with small signaling peptides. EMSA analysis can be used to assess DNA binding of either type of Rgg protein. EMSA analysis of Rgg protein activity has facilitated in vitro confirmation of regulatory targets, identification of precise DNA binding sites via DNA probe mutagenesis, and characterization of the mechanism by which some cognate signaling peptides modulate Rgg protein function (e.g. interruption of DNA-binding in some cases).

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EMSA Analysis of DNA Binding By Rgg Proteins
采用凝胶迁移实验(EMSA)分析Rgg蛋白与DNA的结合

微生物学 > 微生物生物化学 > 蛋白质 > 相互作用
作者: Breah LaSarre
Breah LaSarreAffiliation: Microbiology and Immunology, University of Illinois at Chicago, Chicago, USA
Bio-protocol author page: a507
 and Michael J. Federle
Michael J. FederleAffiliation: Medicinal Chemstry and Pharmacognosy, University of Illionois at Chicago, Chicago, USA
For correspondence: mfederle@uic.edu
Bio-protocol author page: a508
Vol 3, Iss 15, 8/5/2013, 5131 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.838

[Abstract] In bacteria, interaction of various proteins with DNA is essential for the regulation of specific target gene expression. Electrophoretic mobility shift assay (EMSA) is an in vitro approach allowing for the visualization of these protein-DNA interactions. Rgg proteins comprise a family of transcriptional regulators widespread among the Firmicutes. Some of these proteins function independently to regulate target gene expression, while others have now been demonstrated to function as effectors of cell-to-cell communication, having regulatory activitiesthat that are modulated via direct interaction with small signaling peptides. EMSA analysis can be used to assess DNA binding of either type of Rgg protein. EMSA analysis of Rgg protein activity has facilitated in vitro confirmation of regulatory targets, identification of precise DNA binding sites via DNA probe mutagenesis, and characterization of the mechanism by which some cognate signaling peptides modulate Rgg protein function (e.g. interruption of DNA-binding in some cases).

[Abstract]

Materials and Reagents

  1. General chemicals
  2. Non-specific DNA
    e.g. Poly-dIdC (Sigma-Aldrich, catalog number: P4929 )
    Sheared salmon sperm DNA (Amresco, catalog number: E213 )
  3. BSA (10 mg/ml)
  4. Polyacrylamide-bis (40%) for PAGE (e.g. Bio-Rad Laboratories, catalog number: 161-0146 )
  5. Fluorescently-labeled and unlabeled primers for generation of DNA probe (e.g. IDT)
  6. Protein of interest
  7. 4x binding buffer (see Recipes)
  8. 20x running buffer (see Recipes)
  9. 5% Polyacrylamide Gel (see Recipes)

Equipment

  1. Mini-PROTEAN electrophoresis apparatus (Bio-Rad Laboratories)
  2. Fluorescence imaging device (e.g. GE Life Sciences Typhoon PhophorImager)
  3. Mini-PROTEAN casting system (Bio-Rad Laboratories)

Procedure

  1. Procedure for generating fluorescently-tagged DNA probes
    1. Obtain synthetic DNA oligo primers that will be used for PCR amplification of the target DNA of interest (we have had success using primers from IDT). Primers should be designed with the same strategy as general PCR primers (namely a length of 18-30 nt, melting temperatures of both primers in a pair being near 60 °C and within 3 °C of each other, and G + C content between 40%-60%, when possible). At least one of the primers must include a 5'-fluorescent tag (e.g. 6-carboxyfluorescein (6FAM)), but visualization sensitivity can be doubled if both primers are labeled. Generate the fluorescent DNA probe by a standard PCR protocol with any general polymerase enzyme. Probe sizes amenable to EMSA analysis can range from very small (25 bp) to very large (1,000 bp); however, probes between 100-500 bp work well in providing optimal resolution of DNA-protein complexes without requiring extensively long gel running times. If smaller probes are required, the gel percentage may need to be increased or the running time and/or voltage decreased.
    2. Excess oligos and free nucleotides should be separated from the DNA probe using a standard PCR reaction clean-up kit or by gel purifying the product of interest. The DNA probe should be suspended in dH2O or preferred buffer.
    3. The concentration of the probe should be determined using a spectrophotometer. A recommended working concentration is 200 nM (final concentration in EMSA reaction, 10 nM).
    4. Store probes at -20 °C in the dark. Probes can be stored in the manner successfully for several months, although fluorescence intensity may begin to decrease after the first month.

  2. Procedure for purifying Rgg protein of interest
    We have been unable to identify a generic purification scheme compatible with various types of Rgg proteins. Solubility of expressed Rgg proteins varies greatly between homologs, necessitating individual optimization of buffer composition and affinity tag utilization. Examples of successful purification schemes can be found in the methods sections of the references provided below. Regardless of the method of purification, it should be mentioned that it is preferable to generate affinity-tagged Rgg's in such a way as to allow for removal the affinity tag from Rgg prior to use in EMSA assays, thereby helping to ensure that the tag is not interfering with the DNA-binding activity of the protein.

  3. Procedure for EMSA
    1. Cast a 5% non-denaturing polyacrylamide gel following the suggested recipe below. We have successfully used the Bio-Rad mini-PROTEAN casting system for these purposes.
    2. Dilute the Rgg protein to the desired concentration(s) in a solution ensuring its solubility (e.g. storage or binding buffer). The optimal concentration range will depend on the Rgg protein, however serial dilutions resulting in final concentrations of 25-200 nM in the binding reactions should provide a good starting point for such optimization.
    3. Prepare the reaction mixture containing the following components (can prepare master mix of as many components as are shared between all reactions). Dye should not be included in the reaction in order to avoid interference with binding. It should be also noted that control reactions (i.e. probe alone and control probe of DNA not expected to be recognized by the protein) will need to be included as additional lanes on the gel:
      5 μl 4x Binding Buffer + DTT
      X μl non-specific DNA*
      3 μl 80% glycerol
      2 μl 10x BSA*
      1 μl 10 mM CaCl2*
      Y μl dH2O
      18 μl total volume
      Note: The concentrations of the starred components will need to be determined empirically and may be altered or eliminated depending on the activity and DNA-binding specificity of the Rgg protein being used, but the quantities outlined above are good starting points (as is 0.001 U/ml poly-dIdC and 50 μg/ml salmon sperm DNA for non-specific DNA).
    4. To above binding reaction, add 1 μl Rgg protein (or buffer as control).
    5. To each reaction, add 1 μl fluorescently-labeled DNA probe.
    6. Incubate reactions at room temperature (25 °C) for 30 min.
    7. While the reactions are incubating, pre-run gel (no samples) at 100 V, 10 min, 4 °C in 1x running buffer (50 mM potassium phosphate pH 7.5) as the running buffer.
    8. Load 5-10 μl of each reaction on the gel and run at 100 V at 4 °C. The run time should be optimized depending on the size of the DNA probe (for probes between 150-300 bp 60 min is sufficient). Additionally, dye can be run in an empty lane to help visualize migration through the gel (bromophenol blue and xylene cyanol run at approximately 65 nt and 260 nt, respectfully, on a 5% gel).
    9. Transfer the gel to a fluorescence imaging device. We found that sandwiching gels between clear polypropylene sheet protectors was convenient for handling.
      Note: The above binding reaction can be amended to include competitor DNA and/or signaling peptide by reducing the amount of dH2O in the reaction such that the total reaction volume remains 20 μl. Competitor DNA is often included at a concentration of 10-100 fold molar excess relative to the labeled probe, but will need to be optimized for each protein. If competitor DNA is added, add the DNA prior to or simultaneously with the probe. If signaling peptide is included, add the peptide (or empty vehicle as a control) 20 min after the incubation is started (10 min before loading the sample on the gel).

Recipes

  1. 4x binding buffer
    80 mM HEPES (pH 7.9)
    80 mM KCl
    20 mM MgCl2
    0.8 mM EDTA
    2 mM dithiothreitol (added immediately before use)
  2. 20x running buffer
    1 M potassium phosphate (pH 7.5)
    Note: The easiest way to make this is to prepare separate 1 M stocks of both K2HPO4 and KH2PO4, then mix them until the desired pH is reached. Separately, it should be noted that we customarily use potassium phosphate buffer in our EMSAs and have successfully visualized DNA-binding by three different Rgg proteins using this system. However, should it be preferred, it is possible that Tris-based buffer systems would also yield successful results (if the running buffer is changed, the gel buffer will also need to be changed accordingly).
  3. 5% Polyacrylamide Gel (enough for 2 gels; can adjust the gel percentage as needed)
    3.5 ml Acrylamide-bis (40%)
    1 ml 1 M potassium phosphate (pH 7.5)
    16.3 ml dH2O
    200 μl 10% ammonium persulfate (make fresh)
    20 μl TEMED catalyst
    Note: Extra gels can be wrapped in saran wrap or damp paper towels and stored at 4 °C.

References

  1. Chang, J. C., LaSarre, B., Jimenez, J. C., Aggarwal, C. and Federle, M. J. (2011). Two group A streptococcal peptide pheromones act through opposing Rgg regulators to control biofilm development. PLoS Pathog 7(8): e1002190.
  2. LaSarre, B., Aggarwal, C. and Federle, M. J. (2013). Antagonistic Rgg regulators mediate quorum sensing via competitive DNA binding in Streptococcus pyogenes. MBio 3(6): e00333-12.

材料和试剂

  1. 一般化学品
  2. 非特异性DNA
    例如。 Poly-dIdC(Sigma-Aldrich,目录号:P4929)
    剪切的鲑鱼精子DNA(Amresco,目录号:E213)
  3. BSA(10mg/ml)
  4. 用于PAGE的聚丙烯酰胺 - 双(40%)(例如Bio-Rad Laboratories,目录号:161-0146)
  5. 荧光标记和未标记的用于产生DNA探针(例如IDT)的引物
  6. 感兴趣的蛋白质
  7. 4x绑定缓冲区(参见配方)
  8. 20x运行缓冲区(参见配方)
  9. 5%聚丙烯酰胺凝胶(见配方)

设备

  1. Mini-PROTEAN电泳装置(Bio-Rad Laboratories)
  2. 荧光成像装置(例如 GE生命科学台风PhophorImager)
  3. Mini-PROTEAN铸造系统(Bio-Rad Laboratories)

程序

  1. 用于产生荧光标记的DNA探针的程序
    1. 获得将用于感兴趣的目标DNA的PCR扩增的合成DNA寡聚引物(我们已经使用来自IDT的引物成功)。 引物应设计与相同的策略 通常的PCR引物(即长度为18-30nt,一对引物中的两个引物的解链温度在60℃附近且彼此在3℃内,并且如果可能,G + C含量在40%-60%之间) 。至少一种引物必须包括5'-荧光标记(例如6-羧基荧光素(6FAM)),但是如果两种引物都被标记,则可视化灵敏度可以加倍。通过标准PCR方案用任何一般聚合酶产生荧光DNA探针。适合于EMSA分析的探针大小可以从非常小(25bp)到非常大(1000bp);然而,100-500 bp之间的探针能够提供DNA-蛋白复合物的最佳分辨率,而不需要大量长的凝胶运行时间。如果需要更小的探头,凝胶百分比可能需要增加或运行时间和/或电压降低
    2. 应使用标准PCR反应清除试剂盒或通过凝胶纯化目的产物,将过量寡核苷酸和游离核苷酸与DNA探针分离。 DNA探针应悬浮于dH 2 O或优选的缓冲液中
    3. 探针的浓度应使用分光光度计测定。推荐的工作浓度为200nM(在EMSA反应中的最终浓度,10nM)
    4. 将探针储存在-20°C的黑暗中。探针可以成功地存储几个月,但荧光强度可能在第一个月后开始下降
  2. 纯化目的蛋白的方法
    我们已经不能确定与各种类型的Rgg蛋白相容的通用纯化方案。表达的Rgg蛋白的溶解度在同源物之间变化很大,需要个体优化缓冲液组成和亲和标签利用。成功的纯化方案的实例可以在下面提供的参考文献的方法部分中找到。不管纯化方法如何,应该提及的是,优选以这样的方式产生亲和标记的Rgg's,以允许在用于EMSA测定之前从Rgg去除亲和标签,从而帮助确保标签是不干扰蛋白质的DNA结合活性
  3. EMSA程序
    1. 按照以下建议的配方浇铸5%非变性聚丙烯酰胺凝胶。我们已经成功地将Bio-Rad mini-PROTEAN铸造系统用于这些用途
    2. 在溶液中稀释Rgg蛋白至所需浓度,以确保其溶解性(例如,储存或结合缓冲液)。最佳浓度范围将取决于Rgg蛋白,然而在结合反应中导致最终浓度为25-200nM的连续稀释应该为这种优化提供良好的起点。
    3. 制备含有以下组分的反应混合物(可以制备在所有反应之间共享的许多组分的主混合物)。染料不应该包括在反应中以便 避免干扰绑定。还应该注意,需要将包含作为额外泳道的控制反应(即单独的探针和DNA的对照探针不被蛋白质识别)包括在内:
      5μl4x结合缓冲液+ DTT
      Xμl非特异性DNA *
      3微升80%甘油
      2μl10x BSA *
      1μl10mM CaCl 2 *
      YμldH sub 2 O 总体积为18μl
      注意:加星组分的浓度需要根据经验确定,并且可以根据所使用的Rgg蛋白的活性和DNA结合特异性而改变或消除,但上述量是良好的起点(如对于非特异性DNA为0.001U/ml聚dIdC和50μg/ml/ml的鲑精DNA)。
    4. 向上述结合反应中,加入1μlRgg蛋白(或作为对照的缓冲液)
    5. 对每个反应,加入1μl荧光标记的DNA探针
    6. 在室温(25℃)孵育反应30分钟
    7. 当反应在1×运行缓冲液(50mM磷酸钾pH7.5)中作为运行缓冲液在100V,10分钟,4℃下温育预运行凝胶(无样品)。
    8. 在凝胶上加载5-10μl的每个反应,并在4℃下在100V运行。运行时间应根据DNA探针的大小进行优化(对于150-300 bp之间的探针,60分钟就足够了)。此外,染料可以在空白泳道中运行,以帮助观察通过凝胶的迁移(溴苯酚蓝和二甲苯cyanol在约65nt和260nt,分别在5%凝胶上运行)。
    9. 将凝胶转移到荧光成像设备。我们发现,在透明的聚丙烯片保护层之间夹层凝胶是方便的处理 注意:可以通过减少反应中dH 2 O的量来修改上述结合反应以包括竞争物DNA和/或信号肽,使得总反应体积保持20μl。竞争DNA通常以相对于标记探针的10-100倍摩尔过量的浓度包括,但需要对每种蛋白质进行优化。如果加入竞争DNA,在探针之前或同时加入DNA。如果包括信号肽,在温育开始后20分钟(将样品加载到凝胶上之前10分钟)加入肽(或空载体作为对照)。

食谱

  1. 4x结合缓冲液
    80mM HEPES(pH 7.9)
    80 mM KCl
    20mM MgCl 2/
    0.8mM EDTA 2mM二硫苏糖醇(在使用前立即加入)
  2. 20x运行缓冲区
    1M磷酸钾(pH 7.5)
    注意:最简单的方法是制备K sub 2 HPO 4和KH sub 2 PO 4的单独的1M储备液。 sub> 4,然后将它们混合直至达到所需的pH。 另外,应当注意,我们通常在我们的EMSA中使用磷酸钾缓冲液,并且已经使用该系统成功地可视化了通过三种不同的Rgg蛋白的DNA结合。 然而,如果优选,基于Tris的缓冲液系统也可能产生成功的结果(如果运行缓冲液改变,凝胶缓冲液也需要相应地改变)。
  3. 5%聚丙烯酰胺凝胶(足够2凝胶;可根据需要调整凝胶百分比)
    3.5ml丙烯酰胺 - 双(40%) 1ml 1M磷酸钾(pH7.5) 16.3ml dH 2 O·dm / 200μl10%过硫酸铵(新鲜)
    20μlTEMED催化剂
    注意:额外的凝胶可以裹在沙纸或潮湿的纸巾中,并储存在4°C。

参考文献

  1. Chang,J.C.,LaSarre,B.,Jimenez,J.C.,Aggarwal,C.and Federle,M.J。(2011)。 两组A链球菌肽信息素通过相反的Rgg调节剂来控制生物膜的发育。 em> PLoS Pathog 7(8):e1002190。
  2. LaSarre,B.,Aggarwal,C.and Federle,M.J。(2013)。 拮抗性Rgg调节剂通过竞争性DNA结合介导群体感应 化脓性链球菌。 MBio 3(6):e00333-12。
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How to cite this protocol: LaSarre, B. and Federle, M. J. (2013). EMSA Analysis of DNA Binding By Rgg Proteins. Bio-protocol 3(15): e838. DOI: 10.21769/BioProtoc.838; Full Text



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