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Electrophoresis Mobility Shift Assay
电泳迁移率实验

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Abstract

Protein (transcription factors and/or transcription cofactors)-binding to DNA is a critical event in regulation of transcription. Electrophoresis Mobility Shift Assay (EMSA), also known as gel shift assay, is a useful tool to detect protein- or protein complex-DNA/RNA interaction and to evaluate DNA binding specificity of transcription factors in vitro. Here we describe a simple method for EMSA with fluorescent dye-bound oligo DNA probes and recombinant protein expressed in bacterial cells. Using fluorescent dye instead of radioisotope enables easy handling and long-term storage of labelled-probes without reduction of detection sensitivity.

Materials and Reagents

  1. Oligo DNA 5’ end-labeled with IRDye 700 or IRDye 800 (sense strand) (Integrated DNA Technologies)
  2. Non-labelled oligo DNA (both sense and antisense strands)
  3. Non-labelled mutated oligo DNA (both sense and antisense strands)
  4. Recombinant DNA-binding proteins expressed in Escherichia coli (E. coli) (10 ng/µl in protein storage buffer)
  5. Sterile distilled water (SDW)
  6. Odyssey infrared EMSA kit (LI-COR, catalog number: 829-07910 )
  7. Poly(dI-dC) (Sigma-Aldrich, catalog number: 4929 )
  8. Tris
  9. Boric acid
  10. EDTA 2Na
  11. NaCl
  12. HCl
  13. Acrylamide
  14. N,N’-Methylene-bisacrylamide
  15. Glycerol
  16. Triton X-100
  17. Phenylmethylsulfonyl fluoride (PMSF)
  18. β-mercaptoethanol
  19. Ammonium persulfate (APS)
  20. N,N,N',N'-tetramethylethylenediamine (TEMED)
  21. 10x TBE( see Recipes)
  22. 4% native polyacrylamide gel (see Recipes)
  23. Native-PAGE running buffer (see Recipes)
  24. Protein storage buffer (see Recipes)

Equipment

  1. Odyssey CLx Infrared Imaging System (LI-COR)
  2. A set of devices for polyacrylamide gel electrophoresis
  3. Power supply
  4. Refrigerator or cold room
  5. Heat block

Procedure

  1. Suspend lyophilized oligo DNA with dH2O and mix them as follows (final concentration is 50 µM each).
    Probe: labelled and complementary non-labelled oligo DNAs
    Competitor: non-labelled and complementary non-labelled oligo DNAs
    Mutated competitor: mutated non-labelled and complementary non-labelled oligo DNAs
  2. Heat at 100 °C for 10 min on heat block for denature.
  3. Turn off heat block and leave denatured DNAs on the block until room temperature.
  4. Dilute probes to 50 nM with dH2O.
  5. Prepare 4% native polyacrylamide gel and 0.5x TBE.
  6. Pre-electrophoresis for 30 min at 150 V at 4 °C.
  7. Prepare reaction mixtures as follows during pre-electrophoresis:

    Negative Control
    Probe
    Competitor
    Mutated competitor
    Protein (10 ng/µl)
    -
    1 µl
    1 µl
    1 µl
    Probe (50 nM)
    1 µl
    1 µl
    1 µl
    1 µl
    Competitor (50 µM)
    -
    -
    1 µl
    -
    Mutated competitor (50 µM)
    -
    -
    -
    1 µl
    10 x buffer (tube 1)
    2 µl
    2 µl
    2 µl
    2 µl
    25 mM DTT/2.5% Tween-20 (tube 2)
    2 µl
    2 µl
    2 µl
    2 µl
    1 µg/µl Poly (dIdC) (tube 3)
    1 µl
    1 µl
    1 µl
    1 µl
    50% Glycerol (tube 5)
    2 µl
    2 µl
    2 µl
    2 µl
    100 mM MgCl2 (tube 8)
    1 µl
    1 µl
    1 µl
    1 µl
    dH2O
    11 µl
    10 µl
    9 µl
    9 µl
    Note: Tube numbers indicate the vial numbers in Odyssey Infrared EMSA kit.
  8. Place at room temperature for 20 min in dark.
    Note: Avoid light during reaction not to reduce signal intensity.
  9. Add 2 µl of 10x Orange Dye (tube 10) to reaction mixture after reaction.
  10. Wash gel wells with electrophoresis buffer (0.5x TBE) after pre-electrophoresis.
  11. Load the samples on gel and run the gel at 150 V for 2.5 h at 4 °C in dark until the Orange Dye migrates to the bottom of the gel.
  12. Remove gel from glass plate and place it directly on Odyssey.
  13. Adjust focus offset of Odyssey to 1/2 of gel thickness and scan.
    Note: Carefully remove air bubbles between gel and Odyssey.
    Look at supplemental Figure 19 of Reference 1 as a representative EMSA result.

Recipes

  1. 10x TBE
    Consisting of 890 mM Tris, 890 mM boric acid and 20 mM EDTA 2Na (pH 8.3)
    Mix:
    108 g of Tris base
    55 g of boric acid
    3.7 g of EDTA 2Na
    Add dH2O to 1 L
    Autoclave and stored at room temperature
  2. 4% native polyacrylamide gel
    Consisting of 4% acrylamide, 0.5x TBE, 2.5% glycerol, 0.1% APS, 0.1% TEMED
    Mix:
    2.67 ml of 30% acrylamide (acrylamide: bisacrylamide = 29:1)
    1 ml of 10x TBE
    1 ml of 50% glycerol
    0.2 ml of 10% APS
    20 µl of TEMED
    Add dH2O to 20 ml
  3. Native-PAGE running buffer
    0.5x TBE
  4. Protein storage buffer
    Consisting of 10 mM Tris-HCl (pH7.5), 150 mM NaCl, 0.1% Triton X-100, 1 mM PMSF, 0.05% β-mercaptoethanol, 50% glycerol
    Mix:
    1 ml of 1 M Tris-HCl (pH 7.5)
    3 ml of 5 M NaCl
    1 ml of 10% Triton X-100
    1 ml of 100 mM PMSF
    50 µl of β-mercaptoethanol
    50 g of glycerol
    Add dH2O to 100 ml

Acknowledgments

This protocol is adapted from Nakata et al. (2013).

References

  1. Nakata, M., Mitsuda, N., Herde, M., Koo, A. J., Moreno, J. E., Suzuki, K., Howe, G. A. and Ohme-Takagi, M. (2013). A bHLH-type transcription factor, ABA-INDUCIBLE BHLH-TYPE TRANSCRIPTION FACTOR/JA-ASSOCIATED MYC2-LIKE1, acts as a repressor to negatively regulate jasmonate signaling in Arabidopsis. Plant Cell 25(5): 1641-1656.

简介

与DNA结合的蛋白质(转录因子和/或转录辅因子)是调节转录的关键事件。 电泳迁移率变动分析(EMSA),也称为凝胶移位分析,是检测蛋白质或蛋白质复合物-DNA/RNA相互作用和评价转录因子的体外DNA结合特异性的有用工具。 在这里我们介绍一种简单的方法与荧光染料绑定寡DNA探针和表达在细菌细胞中的重组蛋白的EMSA。 使用荧光染料代替放射性同位素使得容易处理和长期储存标记的探针而不降低检测灵敏度。

材料和试剂

  1. 用IRDye 700或IRDye 800(有义链)5'末端标记的寡DNA(整合DNA技术)
  2. 未标记的寡DNA(有义链和反义链)
  3. 非标记的突变寡DNA(有义链和反义链)
  4. 在大肠杆菌中表达的重组DNA结合蛋白(大肠杆菌)(在蛋白质储存缓冲液中为10ng /μl)
  5. 无菌蒸馏水(SDW)
  6. Odyssey红外线EMSA试剂盒(LI-COR,目录号:829-07910)
  7. 聚(dI-dC)(Sigma-Aldrich,目录号:4929)
  8. Tris
  9. 硼酸
  10. EDTA 2Na /
  11. NaCl
  12. HCl
  13. 丙烯酰胺
  14. N,N'-亚甲基 - 双丙烯酰胺
  15. 甘油
  16. Triton X-100
  17. 苯甲基磺酰氟(PMSF)
  18. β-巯基乙醇
  19. 过硫酸铵(APS)
  20. N,N,N',N'-四甲基乙二胺(TEMED)
  21. 10x TBE(参见配方)
  22. 4%天然聚丙烯酰胺凝胶(见配方)
  23. Native-PAGE运行缓冲区(参见配方)
  24. 蛋白质储存缓冲液(见配方)

设备

  1. Odyssey CLx红外成像系统(LI-COR)
  2. 一组用于聚丙烯酰胺凝胶电泳的装置
  3. 电源
  4. 冰箱或冷藏室
  5. 热块

程序

  1. 用dH 2 O 2悬浮冻干的寡DNA,并如下混合(最终浓度为每种50μM)。
    探针:标记的和互补的非标记寡DNA 竞争者:未标记和互补的非标记寡DNA 突变竞争剂:突变的非标记和互补的非标记寡DNA
  2. 在100℃在加热块上加热10分钟以变性
  3. 关闭加热块,将变性的DNA留在块上直到室温
  4. 用dH 2 O稀释探针至50nM
  5. 制备4%的天然聚丙烯酰胺凝胶和0.5×TBE
  6. 在4℃下在150V预电泳30分钟
  7. 在预电泳期间如下制备反应混合物:

    负控制
    探头
    竞争对手
    变异竞争对手
    蛋白(10 ng /μl)
    -
    1微升
    1微升
    1微升
    探针(50 nM)
    1微升
    1微升
    1微升
    1微升
    竞争对手(50μM)
    -
    -
    1微升
    -
    突变竞争者(50μM)
    -
    -
    -
    1微升
    10×缓冲液(试管1)
    2微升
    2微升
    2微升
    2微升
    25mM DTT/2.5%Tween-20(管2) 2微升
    2微升
    2微升
    2微升
    1μg/μlPoly(dIdC)(试管3)
    1微升
    1微升
    1微升
    1微升
    50%甘油(试管5)
    2微升
    2微升
    2微升
    2微升
    100mM MgCl 2(管8)
    1微升
    1微升
    1微升
    1微升
    dH 2 2 O 11微升
    10微升
    9μl
    9μl
    注意:管号表示Odyssey Infrared EMSA套件中的样品瓶编号。
  8. 置于室温下暗处20分钟。
    注意:在反应过程中避免光照,以免降低信号强度。
  9. 在反应后向反应混合物中加入2μl10×橙色染料(管10)
  10. 预电泳后,用电泳缓冲液(0.5×TBE)洗涤凝胶孔
  11. 将样品装载在凝胶上,在4℃下在黑暗中将凝胶在150V下运行2.5小时,直到橙色染料迁移到凝胶底部。
  12. 从玻璃板上取下凝胶,直接放在Odyssey上。
  13. 调整奥德赛的聚焦偏移到凝胶厚度的1/2并扫描 注意:小心地清除凝胶和奥德赛之间的气泡。
    参考参考文献1的补充图19作为代表性的EMSA结果

食谱

  1. 10x TBE
    由890mM Tris,890mM硼酸和20mM EDTA 2Na(pH8.3)组成
    混合:
    108克Tris碱
    55克硼酸 3.7g EDTA 2Na
    将dH <2> O添加到1 L
    高压灭菌并在室温下贮存
  2. 4%天然聚丙烯酰胺凝胶 由4%丙烯酰胺,0.5x TBE,2.5%甘油,0.1%APS,0.1%TEMED组成 混合:
    2.67ml 30%丙烯酰胺(丙烯酰胺:双丙烯酰胺= 29:1) 1 ml 10x TBE
    1ml 50%甘油 0.2ml 10%APS
    20μl的TEMED
    将dH <2> O添加到20ml,
  3. Native-PAGE运行缓冲区
    0.5x TBE
  4. 蛋白质储存缓冲液
    由10mM Tris-HCl(pH7.5),150mM NaCl,0.1%Triton X-100,1mM PMSF,0.05%β-巯基乙醇,50%甘油组成。 混合:
    1ml的1M Tris-HCl(pH7.5) 3ml 5M NaCl
    1ml 10%Triton X-100 1ml 100mM PMSF 50微升β-巯基乙醇 50克甘油 将dH <2> O添加到100 ml

致谢

该协议改编自Nakata等人(2013)。

参考文献

  1. Nakata,M.,Mitsuda,N.,Herde,M.,Koo,A.J.,Moreno,J.E.,Suzuki,K.,Howe,G.A。和Ohme-Takagi, bHLH型转录因子,ABA诱导BHLH型转录因子/JA相关MYC2- LIKE1,作为在拟南芥中负调节茉莉酮酸信号传导的阻遏物。 植物细胞 25(5):1641-1656。
  • 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。
引用:Nakata, M. and Ohme-Takagi, M. (2014). Electrophoresis Mobility Shift Assay. Bio-protocol 4(7): e1099. DOI: 10.21769/BioProtoc.1099.
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