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Liquid Luminescent DNA-precipitation Assay
液相发光DNA沉淀试验   

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Abstract

Working on transcription factors requires studying interactions between protein and DNA. After identification of putative binding-sequences and motifs, Electrophoretic Mobility Shift Assay (EMSA) experiment is classically used to determine specific interactions of proteins and nucleic acids. This lengthy process is rather heavy-handed because of radioisotopically labeled DNA and autoradiographic visualization that are required for the experiments.

Liquid luminescent DNA precipitation assay provides rapid, reliable and quantitative results concerning protein-DNA interactions. This protein-DNA binding assay is based on solution hybridization between Digoxigenin-labeled (DIG) DNA and glutathione S-transferase (GST)-fused DNA binding protein bound to Glutathione Sepharose 4B beads (Figure 1), without electrophoresis (Toshiharu et al., 2008). Digoxigenin is a steroid found in plants. It is increasingly used as a label for nonradioactive detection of nucleic acids and proteins.


Figure 1. Representation of liquid chemiluminescent DNA pull-down assay. A Glutathione S-transferase (GST)-fused NLRP3 (GST-NLRP3) bound to Glutathione Sepharose 4B beads is incubated with a DIG-labeled double-stranded DNA fragment containing putative NLRP3 Binding Site (NBS) in protein-DNA binding buffer. After extensive washing, protein-DNA binding on beads is detected using anti-DIG antibody conjugated to alkaline phosphatase, which is measured by a chemiluminescent reaction using a luminometer Disodium 3-(4-methoxyspiro {1,2-dioxetane-3,2′-(5′-chloro) tricyclo [3.3.1.13, 7] decan}-4-yl) phenyl phosphate(CSPD).

Here, we described how we used this technique to demonstrate the interaction between NLRP3 protein and its DNA binding site (Bruchard et al., 2015).

Keywords: Transcription factor(转录因子), DNA binding(“DNA结合”), Chemiluminescent(化学发光)

Materials and Reagents

  1. BL21 bacteria (DE3) (Thermo Fisher Scientific, InvitrogenTM)
  2. pGEX-4T-1 vector (Addgene, catalog number: 27458001 )
  3. GST fusion NLRP3 protein
  4. Glutathione sepharose 4B (GE Healthcare, catalog number: 17-0756-01 )
  5. Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P9541 )
  6. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S7653 )
  7. Ethylenediaminetetraacetic acid (EDTA), pH 8 (Sigma-Aldrich, catalog number: E9884 )
  8. Dithiothreitol (DTT) (Sigma-Aldrich, catalog number: D0632 )
  9. Glycerol (Sigma-Aldrich, catalog number: G5516 )
  10. Triton X-100 (Sigma-Aldrich, catalog number: T9284 )
  11. Double-stranded oligonucleotides containing the putative binding sequence (Life Technologies)
  12. DIG gel shift kit (Roche Diagnostics, catalog number: 03353591910 )
  13. Disodium 3-(4-methoxyspiro {1, 2-dioxetane-3, 2′-(5′-chloro) tricyclo [3.3.1.13,7] decan}-4-yl) phenyl phosphate (CSPD) (chemiluminescent substrate)
    Note: It is included in DIG gel shift kit.
  14. DNAse/RNase free water (Thermo Fisher Scientific, catalog number: 11538646 )
  15. Tris-HCl (Sigma-Aldrich, catalog number: T5941 )
  16. Binding buffer (see Recipes)
  17. Washing buffer (see Recipes)
  18. Maleic acid buffer (see Recipes)
  19. 10x blocking solution (see Recipes)
  20. Detection buffer (see Recipes)

Equipment

  1. End-over-end rotator
  2. PerkinElmer Envision Plate Reader (PerkinElmer Inc., catalog number: 2104-0010A )

Procedure

GST fusion NLRP3 proteins were previously produced in BL21 bacteria. GST alone was used as a control. Briefly, classical cloning method was used to insert GST-NLRP3 sequence in pGEX-4T-1 vector. BL21 were transformed by a heat shock according to supplier’s instructions. 24 h later bacteria were lysate with 1% Triton X-100 solution.

  1. Day 1
         Binding of GST-fusion protein and Glutathione Sepharose 4B beads
    1. Glutathione Sepharose 4B was washed twice with 5 ml binding buffer and centrifuged at 500 x g for 5 min at room temperature.
    2. GST-fused proteins were purified with Glutathione Sepharose 4B. Three ml of bacteria lysate obtained after BL21 sonication were added to the prepared Glutathione Sepharose 4B in a final volume of 30 ml of binding buffer, and incubated for 30 min at room temperature with gentle agitation. Protein/Glutathione Sepharose 4B complexes were isolated by centrifugation at 500 x g for 5 min at room temperature and washed with 5 ml binding buffer. Centrifugation at 500 x g for 5 min allowed collecting Protein/Glutathione Sepharose 4B complexes.

      DNA labeling reaction
      The NLRP3-binding sequence (NBS) (5′-TCTGTTTTGGGAGGCAGAGCTTTGTTTCTATG-3′) was labeled with Digoxigenin through the use of a DIG Gel Shift Kit but Liquid chemiluminescent DNA pull-down assays can also be performed using biotinylated DNA.
    3. Double-stranded oligonucleotides were diluted to 10 ng/µl with DNAse/RNase free water.
    4. Put the tubes containing 100 ng of double-strand DNA (10 µl) on ice and add 4 µl labeling buffer, 5 mM CoCl2-solution, 0.05 mM DIG-ddUTP solution and 200 U Terminal transferase. Mix by pipetting up and down.
    5. The tubes were centrifuged and incubated for 15 min at 37 °C in a dry block heater and put immediately on ice after this incubation.
    6. The reaction was stopped with 2 µl EDTA.
    7. DNA was then diluted to 4 ng/µl by adding 3 µl of water (DNAse/RNAse free).

      Protein-DNA binding reaction
    8. 2 µg of GST-proteins bound to Glutathione Sepharose 4B beads were incubated with 20 fmol DIG-labeled double-stranded DNA fragments in protein-DNA binding buffer from the DIG Gel Shift Kit in a final volume of 20 µl overnight at room temperature.

  2. Day 2
    1. GST-fused proteins bound to DNA were washed three times with 1 ml Washing Buffer from the kit and centrifuged each time at 500 x g for 5 min at room temperature.
    2. Then the beads were incubated in 500 µl 1x blocking solution (dilution of 10x blocking solution in maleic acid buffer) for 30 min at room temperature.
    3. GST-fused proteins bound to DNA were incubated with anti-DIG Fab fragments conjugated with 75 mU/ml alkaline phosphatase (from DIG gel shit kit) for 30 min at room temperature in 1x blocking solution.
    4. GST-fused proteins bound to DNA are washed twice with 5 ml Washing Buffer.
    5. After washes, the complexes were transferred in detection buffer to 96-well plates and were incubated for 5 min at room temperature with 1 μg/ml 3-(4-methoxyspiro {1,2-dioxetane-3,2′-(5′-chloro) tricyclo [3.3.1.13,7] decan}-4-yl) phenyl phosphate (CSPD) , followed by incubation for 10 min at 37 °C.
    6. Light emission was measured using a PerkinElmer Envision Plate Reader. Light detection was performed during 1 sec per well.

Representative data

The emission signal from all the reagents mixed together without DNA-DIG was used for normalization and data are presented as arbitrary units. All experimental conditions were tested with GST-NLRP3 and GST alone. Double strand DNA labeled without DIG was tested alone and used as a competitor (Figure 2).


Figure 2. Representative data of liquid chemiluminescent DNA pull-down assay. Light emission was measured from the liquid chemiluminescent DNA pull-down assay to indicate binding. The histograms indicate relative light emission (arbritary units, AU). Glutathione S-transferase (GST)-NLRP3 was incubated with Digoxigenin (DIG)-labeled NBS with or without Competitor (non labelled NBS). Data are shown as means ± SEM.

Recipes

  1. Binding buffer
    75 mM KCl
    50 mM NaCl
    1 mM EDTA
    1 mM DTT
    10% glycerol
    0.1% Triton X-100
  2. Washing buffer
    0.1 M maleic acid
    0.15 M NaCl (pH 7.5)
    0.3% (v/v) Tween 20
  3. Maleic acid buffer
    0.1 M maleic acid
    0.15 M NaCl
    Adjust with NaOH to pH 7.5
  4. 10x blocking solution
    10% (w/v) blocking reagent in maleic acid buffer
  5. Detection buffer
    0.1 M Tris-HCl
    0.1 M NaCl (pH 9.5)

Acknowledgments

This protocol is adapted from Toshiharu et al. (2008). This project was supported by the French National Research Agency (“Investissements d’Avenir” program; ANR-11-LABX-0021), the Ligue nationale contre le cancer (F. G. and F. V.), the Institut National du Cancer (F. G.), Fondation pour la Recherche Médicale (F. G).

References

  1. Bruchard, M., Rebe, C., Derangere, V., Togbe, D., Ryffel, B., Boidot, R., Humblin, E., Hamman, A., Chalmin, F., Berger, H., Chevriaux, A., Limagne, E., Apetoh, L., Vegran, F. and Ghiringhelli, F. (2015). The receptor NLRP3 is a transcriptional regulator of TH2 differentiation. Nat Immunol 16(8): 859-870.
  2. Iwasaki, T., Miyazaki, W., Rokutanda, N. and Koibuchi, N. (2008). Liquid chemiluminescent DNA pull-down assay to measure nuclear receptor-DNA binding in solution. Biotechniques 45(4): 445-448.

简介

研究转录因子需要研究蛋白质和DNA之间的相互作用。在推定的结合序列和基序的鉴定之后,电泳迁移率变动分析(EMSA)实验通常用于确定蛋白质和核酸的特异性相互作用。这个漫长的过程相当沉重,因为放射性同位素标记的DNA和实验所需的自动放射成像可视化。
 液体发光DNA沉淀测定提供关于蛋白质-DNA相互作用的快速,可靠和定量的结果。这种蛋白质-DNA结合测定基于与洋地黄毒苷标记的(DIG)DNA和结合至谷胱甘肽Sepharose 4B珠粒的谷胱甘肽S-转移酶(GST)融合的DNA结合蛋白(图1)之间的溶液杂交,而不进行电泳(Toshiharu, et al。,,2008)。地高辛是在植物中发现的类固醇。它越来越多地用作核酸和蛋白质的非放射性检测的标签。



图1.液体化学发光DNA下拉试验的表示。谷胱甘肽转移酶(GST)将与谷胱甘肽Sepharose 4B珠结合的融合NLRP3(GST-NLRP3)与在蛋白质-DNA结合缓冲液中含有推定的NLRP3结合位点(NBS)的DIG标记的双链DNA片段孵育。在广泛洗涤后,使用缀合碱性磷酸酶的抗DIG抗体检测珠上的蛋白质-DNA结合,其通过使用发光计的化学发光反应测量3-(4-甲氧基螺[1,2-二氧杂环丁烷-3,2' - (5'-氯)三环[3.3.1.1] 3,7]癸烷-4-基)苯基磷酸盐(CSPD)。

 在这里,我们描述了我们如何使用这种技术来证明NLRP3蛋白与其DNA结合位点之间的相互作用(Bruchard等人,2015)。

关键字:转录因子, “DNA结合”, 化学发光

材料和试剂

  1. BL21细菌(DE3)(Thermo Fisher Scientific,Invitrogen TM
  2. pGEX-4T-1载体(Addgene,目录号:27458001)
  3. GST融合NLRP3蛋白
  4. 谷胱甘肽琼脂糖4B(GE Healthcare,目录号:17-0756-01)
  5. 氯化钾(KCl)(Sigma-Aldrich,目录号:P9541)
  6. 氯化钠(NaCl)(Sigma-Aldrich,目录号:S7653)
  7. 乙二胺四乙酸(EDTA),pH 8(Sigma-Aldrich,目录号:E9884)
  8. 二硫苏糖醇(DTT)(Sigma-Aldrich,目录号:D0632)
  9. 甘油(Sigma-Aldrich,目录号:G5516)
  10. Triton X-100(Sigma-Aldrich,目录号:T9284)
  11. 含有推定的结合序列的双链寡核苷酸(Life Technologies)
  12. DIG凝胶移位试剂盒(Roche Diagnostics,目录号:03353591910)
  13. 3-(4-甲氧基螺[1,2] - 二氧杂环丁烷-3,2' - (5'-氯)三环[3.3.1.1 3,7]癸烷-4-基)苯基磷酸二钠 (CSPD)(化学发光底物)
    注意:它包含在DIG凝胶移位套件中。
  14. DNAse/RNAse游离水(Thermo Fisher Scientific,目录号:11538646)
  15. Tris-HCl(Sigma-Aldrich,目录号:T5941)
  16. 绑定缓冲区(参见配方)
  17. 洗涤缓冲液(见配方)
  18. 马来酸缓冲液(参见配方)
  19. 10x封锁溶液(参见配方)
  20. 检测缓冲区(参见配方)

设备

  1. 端到端旋转器
  2. PerkinElmer Envision Plate Reader(PerkinElmer Inc.,目录号:2104-0010A)

程序

GST融合NLRP3蛋白以前在BL21细菌中产生。 单独的GST用作对照。 简言之,使用经典克隆方法将GST-NLRP3序列插入pGEX-4T-1载体中。 BL21根据供应商的说明通过热休克转化。 24小时后,用1%Triton X-100溶液裂解细菌。

  1. 第1天
         GST-融合蛋白和谷胱甘肽Sepharose 4B珠的结合
    1. 将谷胱甘肽琼脂糖4B用5ml结合缓冲液洗涤两次并在室温下以500×g离心5分钟。
    2. GST融合蛋白用谷胱甘肽琼脂糖4B纯化。 加入在BL21超声处理后获得的3ml细菌裂解物制备的谷胱甘肽琼脂糖4B,最终体积为30ml 结合缓冲液,并在室温下温和温育30分钟  搅动。通过分离蛋白/谷胱甘肽Sepharose 4B复合物 在室温下以500×g离心5分钟,并用5℃洗涤  ml结合缓冲液。在500×g离心5分钟 收集蛋白/谷胱甘肽Sepharose 4B复合物
      DNA标记反应
      NLRP3结合序列(NBS) (5'-TCTGTTTTGGGAGGCAGAGCTTTGTTTCTATG-3')用洋地黄毒苷 通过使用DIG凝胶移位试剂盒,但液体化学发光DNA 也可以使用生物素化的DNA进行下拉测定
    3. 将双链寡核苷酸用不含DNAse/RNAse的水稀释至10ng /μl。
    4. 将含有100ng双链DNA(10μl)的试管置于冰上 并加入4μl标记缓冲液,5mM CoCl 2溶液,0.05mM DIG-ddUTP溶液和200U末端转移酶。 通过上下吹吸混合。
    5. 将管离心并在37℃下孵育15分钟 干块加热器,并在孵育后立即置于冰上
    6. 用2μlEDTA终止反应
    7. 然后通过加入3μl水(无DNAse/RNAse)将DNA稀释至4ng /μl
      蛋白质-DNA结合反应
    8. 将2μg与谷胱甘肽Sepharose 4B珠结合的GST-蛋白 与20fmol DIG标记的双链DNA片段孵育 蛋白质-DNA结合缓冲液从DIG Gel Shift Kit在最终体积 的20μl在室温下过夜

  2. 第2天
    1. 用1ml将与DNA结合的GST融合蛋白洗涤3次 洗涤缓冲液,并在500×g下每次离心5分钟 min。
    2. 然后将珠子在500μl中温育   1x封闭溶液(在马来酸中稀释10倍封闭溶液 缓冲液)在室温下处理30分钟
    3. GST融合蛋白结合与与75缀合的抗DIG Fab片段一起孵育 mU/ml碱性磷酸酶(来自DIG凝胶试剂盒)在室温下30分钟 温度在1x封闭溶液中
    4. 与DNA结合的GST-融合蛋白用5ml洗涤缓冲液洗涤两次
    5. 洗涤后,将复合物转移到检测缓冲液中   96孔板中,并在室温下与1℃孵育5分钟 μg/ml 3-(4-甲氧基螺[1,2-二氧杂环丁烷-3,2' - (5'-氯)三环 [3.3.1.1] 3,7]癸烷} -4-基)苯基磷酸酯(CSPD),随后 在37℃孵育10分钟
    6. 使用PerkinElmer Envision Plate Reader测量光发射。 在每孔1秒内进行光检测。

代表数据

来自所有混合在一起而没有DNA-DIG的试剂的发射信号用于标准化,数据以任意单位表示。所有实验条件用单独的GST-NLRP3和GST测试。单独测试不带DIG标记的双链DNA,并用作竞争剂(图2)

图2.液体化学发光DNA下拉测定的代表性数据。从液体化学发光DNA下拉测定中测量光发射,以指示结合。直方图指示相对光发射(arbritary units,AU)。谷胱甘肽-S-转移酶(GST)-NLRP3与洋地黄毒苷(DIG)标记的NBS一起温育,有或没有竞争剂(未标记的NBS)。数据显示为平均值±sem。

食谱

  1. 绑定缓冲区
    75 mM KCl
    50mM NaCl 1mM EDTA
    1 mM DTT
    10%甘油 0.1%Triton X-100
  2. 洗涤缓冲液
    0.1M马来酸
    0.15 M NaCl(pH 7.5)
    0.3%(v/v)吐温20
  3. 马来酸缓冲液
    0.1M马来酸
    0.15 M NaCl
    用NaOH调节至pH7.5
  4. 10x封闭溶液
    10%(w/v)封闭剂在马来酸缓冲液中
  5. 检测缓冲区
    0.1M Tris-HCl
    0.1 M NaCl(pH 9.5)

致谢

该协议改编自Toshiharu等人 (2008)。 该项目得到了法国国家研究机构("Investissements d'Avenir"方案; ANR-11-LABX-0021),国家癌症研究所(FG和FV),国家癌症研究所 pour la RechercheMédicale(F. G)。

参考文献

  1. Bruchard,M.,Rebe,C.,Derangere,V.,Togbe,D.,Ryffel,B.,Boidot,R.,Humblin,E.,Hamman,A.,Chalmin,F.,Berger, Chevriaux,A.,Limagne,E.,Apetoh,L.,Vegran,F.and Ghiringhelli,F.(2015)。 受体NLRP3是TH2分化的转录调节子。 Nat Immunol 16(8):859-870。
  2. Iwasaki,T.,Miyazaki,W.,Rokutanda,N.and Koibuchi,N。(2008)。 液体化学发光DNA拉下测定法,以测量溶液中的核受体-DNA结合。 Biotechniques 45(4):445-448。
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
引用:Végran, F., Bruchard, M., Derangère, V. and Ghiringhelli, F. (2016). Liquid Luminescent DNA-precipitation Assay. Bio-protocol 6(10): e1812. DOI: 10.21769/BioProtoc.1812.
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