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A Protocol for Electrophoretic Mobility Shift Assay (EMSA) from Primary Neuron
原代神经元的电泳迁移改变分析(EMSA)方法   

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Abstract

The interaction of transcriptional or co-transcriptional factors with DNA is crucial for changes of neuronal gene expression during normal brain development as well as neurodegeneration. The electrophoretic mobility shift assay (EMSA) is a very powerful technique for studying changes of neuronal gene expression and determining protein: DNA interactions. EMSA can be used qualitatively to identify specific transcriptional or co-transcriptional factors in brain crude lysates or primary neurons and, in conjunction with mutagenesis, to identify the important binding sequences within a given gene. An advantage of studying protein: DNA interaction by an electrophoretic assay provides a better understanding of epigenetic changes during normal brain development and neurodegenerative process.

Materials and Reagents

  1. Neuronal cell pellet
  2. Biotin 5' end-labeled and non-labeled DNA probes (competition) (Integrated DNA Technologies)
    Table S- sequences of EMSA probes


  3. Positively charged nylon membrane (Sigma-Aldrich, catalog number: Z670197 )
  4. Tris base
  5. Boric acid
  6. EDTA
  7. BSA
  8. Poly (dIdC) (0.5 μg/μl) (Pierce Antibodies, catalog number: 20148 )
  9. Antibody (ab1437)
  10. 5x loading buffer (QIAGEN, catalog number: 1037650 )
  11. X-ray film
  12. High-quality blotting paper (Whatman, catalog number: 3030-931 )
  13. Polyacrylamide gel in 0.5x TBE
  14. Cytoplasmic extract buffer (NE-PER Nuclear and Cytoplasmic Extraction Kit) (Pierce Antibodies, catalog number: 78835 )
  15. Washing buffer (LightShift Chemiluminescent EMSA Kit) (Pierce Antibodies, catalog number: 20148)
  16. Nuclear extraction buffer (NE-PER Nuclear and Cytoplasmic Extraction Kit) (Pierce Antibodies, catalog number: 78835 )
  17. 2x reaction buffer (LightShift Chemiluminescent EMSA Kit) (Pierce Antibodies, catalog number: 20148)
  18. Acrylamide
  19. Bis-acrylamide
  20. TBE buffer
  21. TEMED
  22. Ammonium persufate
  23. Phosphatase inhibitors
  24. 6% non-denature PAGE gel (see Recipes)
  25. 5x TBE (pH 8.3) (see Recipes)

Equipment

  1. Centrifuges
  2. UV lamp or crosslinking device equipped with 254 nm bulbs or 312 nm transilluminator
  3. Electrophoresis apparatus
  4. Electroblotter or capillary transfer apparatus
  5. 1.5 ml microcentrifuge tube

Procedure

  1. Prepare the nuclear protein extract from neuron
    1. Collect 1-5 x 106-7 neurons pellet in 1.5 ml microcentrifuge tube (DIV 14 Primary cultural neuron was from mouse E16.5 cortex).
    2. Resuspend neurons in 200 μl cytoplasmic extract buffer with protease and phosphatase inhibitors, and keep on ice for 10 min to break the cell membrane.
    3. Spin cells at 16,000 x g for 5 min at 4 °C to separate nuclei with cytoplasmic component (quality of fraction was tested by nuclear/cytoplasmic protein markers-HDAC1 and HSP90).
    4. Remove supernatant as cytoplasmic extract.
    5. Wash the nuclear pellet with 300 μl washing buffer.
    6. Spin cells at 16,000 x g for 10 min at 4 °C to pellet the nuclei.
    7. Resuspend the nuclear pellet in 100 μl nuclear extraction buffer and aliquot the lysate into10 μl/tube.
    8. Freeze the nuclear extracts in -80 °C.
    9. Measure protein concentration and adjust it to 1 μg/μl with the extraction buffer for use in gel shift assay.

  2. Prepare and pre-run gel
    1. Prepare a native PAGE gel in 0.5x TBE. The appropriate polyacrylamide percent depends on the size of the target DNA and the binding protein. Most systems use a 4 -6% PAGE gel in 0.5x TBE.
    2. Place the gel in the electrophoresis unit. Fill the inner chamber with 0.5x TBE to a height several millimeters above the top of the wells. Fill the outside of the tank with 0.5x TBE to just above the bottom of the wells, which reduces heat during electrophoresis. Flush wells and pre-electrophorese the gel for 30-60 min at 100 V.

  3. Perform binding reactions
    1. 2x reaction buffer 12 μl.
    2. BSA (1 μg/μl) 3 μl.
    3. Poly (dIdC) (0.5 μg/μl) 2 μl.
    4. Nuclear extract (1 μg/μl) 3 μl (cytoplasmic extract as control).
    5. dH2O 3 μl.
    6. Keep at room temperature or on ice for 10 min without Antibody, 20 min with Antibody; Keep rotation (option).
    7. Add in Biotin-labeled DNA probe (20 fmol/ reaction).
    8. Keep at room temperature for 20 min.

  4. Electrophorese binding reactions
    1. Add 5 μl of 5x loading buffer to each 20 μl binding reaction, pipetting up and down several times to mix. Run gel at 200 V for 1-1.5 h. Use DNA loading buffer in lane 1 as indicator of free probe. Free probe usually run at the same mobility as the blue dye of the DNA loading buffer. Stop the gel when the dye runs at 3 cm to the bottom.

  5. Electrophoretic transfer of binding reactions to nylon membrane
    1. Soak nylon membrane in 0.5x TBE for 15 min.
    2. Sandwich the gel, nylon membrane and blotting paper in a clean electrophoretic transfer unit.
    3. Transfer at 380 mA (~100 V) for 30 min.
    4. When the transfer is complete, place the on a dry paper towel for 1-3 min.

  6. Crosslink transferred DNA to membrane and detection
    1. 10-15 min with the membrane face down on a transilluminator equipped with 312 nm bulbs.
    2. After the membrane is crosslinked, proceed directly to the LightShift Chemiluminescent detection kit. Alternatively, the membrane may be stored dry at room temperature for several days.

Recipes

  1. 6% non-denature PAGE gel
    For example, to make 50 ml volumes gel:
    7.5ml 40% acrylamide
    5ml 2% Bis-acrylamide
    2.5ml 10x TBE buffer
    50 μl TEMED
    0.5 ml 10% ammonium persufate
    34.5 ml dH2O
  2. 5x TBE (pH 8.3)
    450 mM Tris
    450 mM boric acid
    10 mM EDTA

Acknowledgments

This protocol is adapted from Li et al. (2012).

References

  1. Li, J., Chen, J., Ricupero, C. L., Hart, R. P., Schwartz, M. S., Kusnecov, A. and Herrup, K. (2012). Nuclear accumulation of HDAC4 in ATM deficiency promotes neurodegeneration in ataxia telangiectasia. Nat Med 18(5): 783-790.

简介

转录或共转录因子与DNA的相互作用对于正常脑发育以及神经变性过程中神经元基因表达的变化是至关重要的。 电泳迁移率变动分析(EMSA)是一种非常强大的技术,用于研究神经元基因表达的变化和确定蛋白质:DNA相互作用。 EMSA可定性地用于鉴定脑粗提裂解物或原代神经元中的特定转录或共转录因子,并且与诱变相结合以鉴定给定基因内的重要结合序列。 通过电泳测定研究蛋白质:DNA相互作用的优点提供了对正常脑发育和神经变性过程中的表观遗传改变的更好理解。

材料和试剂

  1. 神经元细胞沉淀
  2. 生物素5'端标记和未标记的DNA探针(竞争)(Integrated DNA Technologies)
    表S- EMSA探针的序列


  3. 带正电荷的尼龙膜(Sigma-Aldrich,目录号:Z670197)
  4. Tris碱
  5. 硼酸
  6. EDTA
  7. BSA
  8. Poly(dIdC)(0.5μg/μl)(Pierce Antibodies,目录号:20148)
  9. 抗体(ab1437)
  10. 5x加样缓冲液(QIAGEN,目录号:1037650)
  11. X光片
  12. 高品质吸墨纸(Whatman,目录号:3030-931)
  13. 聚丙烯酰胺凝胶在0.5×TBE中
  14. 细胞质提取缓冲液(NE-PER核和细胞质提取试剂盒)(Pierce Antibodies,目录号:78835)
  15. 洗涤缓冲液(LightShift Chemiluminescent EMSA试剂盒)(Pierce Antibodies,目录号:20148)
  16. 核提取缓冲液(NE-PER核和细胞质提取试剂盒)(Pierce Antibodies,目录号:78835)
  17. 2x反应缓冲液(LightShift Chemiluminescent EMSA试剂盒)(Pierce Antibodies,目录号:20148)
  18. 丙烯酰胺
  19. 双丙烯酰胺
  20. TBE缓冲区
  21. TEMED
  22. 过硫酸铵
  23. 磷酸酶抑制剂
  24. 6%非变性PAGE凝胶(见配方)
  25. 5x TBE(pH 8.3)(参见配方)

设备

  1. 离心机
  2. UV灯或装备有254nm灯泡或312nm透射仪的交联装置
  3. 电泳仪
  4. 电凝器或毛细管转移装置
  5. 1.5 ml微量离心管

程序

  1. 准备神经元的核蛋白提取物
    1. 在1.5ml微量离心管(DIV14原代培养神经元来自小鼠E16.5皮层)中收集1-5×10 6个 神经元沉淀。
    2. 重悬浮神经元在200微升细胞质提取缓冲液中的蛋白酶和磷酸酶抑制剂,并保持在冰上10分钟打破细胞膜。
    3. 在4℃下以16,000×g离心5分钟以分离具有胞质组分的细胞核(通过核/细胞质蛋白标记物-HDAC1和HSP90测试级分质量)。
    4. 除去上清液作为细胞质提取物
    5. 用300μl洗涤缓冲液洗涤核沉淀。
    6. 在4℃下将细胞以16,000×g离心10分钟以沉淀核。
    7. 将核沉淀重悬在100μl核提取缓冲液中,将裂解物等分到10μl/管中
    8. 在-80℃下冻干核提取物。
    9. 测量蛋白质浓度,并用提取缓冲液调整至1μg/μl,用于凝胶迁移测定

  2. 准备和预运行凝胶
    1. 在0.5x TBE中制备天然PAGE凝胶。 合适的聚丙烯酰胺百分比取决于靶DNA和结合蛋白的大小。 大多数系统在0.5x TBE中使用4 -6%PAGE凝胶
    2. 将凝胶放置在电泳单元中。 用0.5×TBE填充内室至高于孔顶部几毫米的高度。 将0.5x TBE的罐外部填充到孔底部,这样可以减少电泳过程中的热量。 冲洗孔并在100V下预凝胶电泳30-60分钟。

  3. 执行结合反应
    1. 2×反应缓冲液12μl
    2. BSA(1μg/μl)3μl
    3. Poly(dIdC)(0.5μg/μl)2μl
    4. 核提取物(1μg/μl)3μl(作为对照的细胞质提取物)
    5. dH 2 O3μl。
    6. 保持在室温或在冰上10分钟无抗体,20分钟与抗体; 继续旋转(选项)。
    7. 加入生物素标记的DNA探针(20fmol /反应)
    8. 在室温下保持20分钟。

  4. 电泳结合反应
    1. 向每个20μl结合反应中加入5μl5x上样缓冲液,上下吹打数次混匀。 在200V运行凝胶1-1.5小时。 使用泳道1中的DNA上样缓冲液作为游离探针的指示剂。 游离探针通常以与DNA上样缓冲液的蓝色染料相同的迁移率运行。 当染料在距离底部3cm处停止时停止凝胶。

  5. 尼龙膜结合反应的电泳转移
    1. 在0.5×TBE中浸泡尼龙膜15分钟
    2. 在干净的电泳转移装置中将凝胶,尼龙膜和吸墨纸夹在中间
    3. 在380mA(〜100V)下转移30分钟。
    4. 当转印完成后,将其放在干纸巾上1-3分钟。

  6. 交联将DNA转移到膜和检测
    1. 10-15分钟,膜面向下放在装有312nm灯泡的透照器上
    2. 膜交联后,直接进入LightShift Chemiluminescent检测试剂盒。 或者,膜可以在室温下干燥储存几天

食谱

  1. 6%非变性PAGE凝胶 例如,使50ml体积凝胶:
    7.5ml 40%丙烯酰胺 5ml 2%双丙烯酰胺 2.5ml 10x TBE缓冲液
    50μlTEMED
    0.5ml 10%过硫酸铵 34.5ml dH 2 O 2 /
  2. 5x TBE(pH 8.3)
    450 mM Tris
    450mM硼酸 10 mM EDTA

致谢

该协议改编自Li等人(2012)。

参考文献

  1. Li,J.,Chen,J.,Ricupero,C.L.,Hart,R.P.,Schwartz,M.S.,Kusnecov,A.and Herrup,K。(2012)。 HDAC4在ATM缺陷中的核积累可促进共济失调毛细血管扩张症中的神经变性。 Med 18(5):783-790。
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Copyright: © 2012 The Authors; exclusive licensee Bio-protocol LLC.
引用:Li, J. (2012). A Protocol for Electrophoretic Mobility Shift Assay (EMSA) from Primary Neuron. Bio-protocol 2(23): e300. DOI: 10.21769/BioProtoc.300.
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Britney Helling
UC Denver
Hi,
What is the purpose of the crosslinking and can I get away with not doing it if I image it directly after chemiluminescence?
Thanks
7/27/2015 4:33:30 PM Reply
Randi Foxall
University of New Hampshire
I am trying to troubleshoot an EMSA and came across your protocol. I am performing an EMSA using 5' biotin ssDNA, not dsDNA as in your protocol. Do you know if a ssDNA probe should work with the detection assays for Biotin?
9/5/2014 10:15:26 AM Reply
Jiali Li
Department of Cell Biology and Neuroscience, Nelson Biological Laboratories, Rutgers University, USA

It should work too if it binds to your target efficiently.

9/6/2014 12:00:39 AM


pritha ray
ACTREC, Tata memorial Centre
Hi,
Thanks for the protocol. In my experiment i am not seeing any trace of the free labeled probe at the bottom of the gel though a retarded band is present at high molecular weight. This band can be competed out with excess cold probe. I tried running a very short gel by keeping bromophenol blue at 1/3rd of the gel and still didnot see any free probe. The size of the oligo is 50 bp. Please suggest.
7/19/2013 5:08:48 AM Reply
Jiali Li
Department of Cell Biology and Neuroscience, Nelson Biological Laboratories, Rutgers University, USA

Please make sure whether a correct DNA-running gel is used. Is any positive control set up?

7/19/2013 6:21:22 AM