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[Bio101] A General EMSA (Gel-shift) Protocol
[Bio101] 凝胶阻滞实验(EMSA)   

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Abstract

An electrophoretic mobility shift assay (EMSA), also referred to as mobility shift electrophoresis, a gel shift assay, gel mobility shift assay, band shift assay, or gel retardation assay, is a common technique used to study protein-DNA or protein-RNA interactions. The control lane (the DNA/RNA probe without protein present) will contain a single band corresponding to the unbound DNA or RNA fragment. If the protein is capable of binding to the fragment, the lane with protein present will contain another band that represents the larger, less mobile complex of nucleic acid probe bound to the protein, which is 'shifted' up on the gel (since it has moved more slowly). Here, a protocol to carry out an EMSA assay is described.

Keywords: EMSA(EMSA), Gel-shift(凝胶移位), Binding(结合)

Materials and Reagents

  1. DTT (Promega Corporation, catalog number: V3151 )
  2. Poly-dIdC (Sigma-Aldrich, catalog number: P4929-10UN )
  3. 32P-labeled probe
    Note: Oligo DNA probe can be synthesized ordered from IDT, a DNA synthesis company, then labeled by yourself.
  4. BSA (Sigma-Aldrich, catalog number: 05470-5G )
  5. General chemicals (Sigma-Aldrich)
  6. 1.5x binding buffer (see Recipes)
  7. 10x TBE buffer (see Recipes)

Equipment

  1. Plates
  2. Spacers
  3. Clamps
  4. Saran wrap
  5. Whatman paper (GE Healthcare)

Procedure

  1. Pour protein polyacrylamide gel.
    1. Assemble plates, spacers, and clamps. Seal with 1% agarose to prevent leaks.
    2. Pour 5% polyacrylamide gel.
      Plate size
      Large
      Medium
      H2O
      78 ml
      39 ml
      10x TBE
      5 ml
      2.5 ml
      30% acrylamide stock (19:1)
      16.6 ml
      8.4 ml
      10% APS
      1,000 μl
      500 μl
      Mix well while minimizing bubble formation. Add 100 μl/50 μl TEMED. Mix and pour, add combs. Gel will take ~10 min to polymerize. After polymerization, gel can be wrapped in saran wrap and stored at 4 °C.
  2. Prepare 5x binding buffer.
  3. Set binding reaction:
    1 μl of poly-dIdC (1 μg/μl in TE)
    2 μl of 5x binding buffer
    1 μl of labeled probe
    1 μl cold competitor - unlabeled DNA fragments containing the binding sequences (if needed)
    0.1 μl 100x BSA
    X μl nuclear extract (5 μg protein total)
    Add H2O to 10 μl final volume
    Incubate for 30 min at room temperature (RT). Add antibody for supershift (if needed). Incubate additional 30 min at RT.
  4. While binding reaction is incubating, run the polyacrylamide gel without any sample at 150 V, 30 min, using 0.5x TBE as the running buffer. Then run samples on the polyacrylamide gel for ~2 h at 150 V.
  5. Dry the gel (optional).
    Transfer gel to Whatman paper. Cover top of gel with saran wrap and dry at 80 °C in vacuum dryer for 1-2 h.
  6. Expose the gel.
    Place gel in cassette with reflection screen. Add film and place in -80 °C freezer.

Recipes

  1. 5x binding buffer
    Composition
    Recipe for 10 ml
    50 mM Tris HCl (pH 8.0)
    0.5 ml of 1 M Tris HCl (pH 8.0)
    750 mM KCl
    3 ml of 2.5 M KCl
    2.5 mM EDTA
    50 μl of 0.5 M EDTA (pH 8.0)
    0.5% Triton-X 100
    50 μl Triton-X 100
    62.5 % glycerol (v/v)
    7.87 g glycerol
    1 mM DTT
    add DTT fresh before use
  2. 10x TBE buffer (1 L)
    106 g of Tris base
    55 g of boric acid
    40 ml of 0.5 M EDTA (pH 8.0)

Acknowledgments

This work was funded by 5050 project by Hangzhou Hi-Tech District, Funding for Oversea Returnee by Hangzhou City, ZJ1000 project by Zhejiang Province. The protocol was developed in the Cohen Lab, Department of Genetics, Stanford University, CA, USA.

References

  1. Chen, R., Liliental, J. E., Kowalski, P. E., Lu, Q. and Cohen, S. N. (2011). Regulation of transcription of hypoxia-inducible factor-1alpha (HIF-1alpha) by heat shock factors HSF2 and HSF4. Oncogene 30(22): 2570-2580.

简介

电泳迁移率变动测定(EMSA),也称为迁移率变动电泳,凝胶迁移测定,凝胶迁移率变动测定,带移测定或凝胶阻滞测定是用于研究蛋白质-DNA或蛋白质-RNA的常见技术 互动。 对照泳道(不含蛋白质的DNA/RNA探针)将含有对应于未结合的DNA或RNA片段的单个条带。 如果蛋白质能够结合片段,存在蛋白质的泳道将包含另一条带,其代表与蛋白质结合的核酸探针的较大的,较少移动的复合物,其在凝胶上"移位"(因为它具有 移动更慢)。 这里,描述了进行EMSA测定的方案。

关键字:EMSA, 凝胶移位, 结合

材料和试剂

  1. DTT(Promega Corporation,目录号:V3151)
  2. Poly-dIdC(Sigma-Aldrich,目录号:P4929-10UN)
  3. 32 P标记的探针
    注意:Oligo DNA探针可以从DNA合成公司IDT订购,然后自己标记。
  4. BSA(Sigma-Aldrich,目录号:05470-5G)
  5. 一般化学品(Sigma-Aldrich)
  6. 1.5x绑定缓冲区(参见配方)
  7. 10x TBE缓冲区(参见配方)

设备


  1. 间隔符
  2. 夹具
  3. Saran换装
  4. Whatman纸(GE Healthcare)

程序

  1. 倒蛋白聚丙烯酰胺凝胶。
    1. 组装板,间隔件和夹具。 用1%琼脂糖密封以防止泄漏
    2. 倒入5%聚丙烯酰胺凝胶。
      板尺寸

      中等
      H sub 2 O
      78 ml
      39 ml
      10x TBE
      5 ml
      2.5 ml
      30%丙烯酰胺储液(19:1) 16.6 ml
      8.4 ml
      10%APS
      1000微升
      500微升
      混合良好,同时最小化气泡形成。 加入100μl/50μlTEMED。 混合并倒入,加入梳子。 凝胶需要〜10分钟聚合。 聚合后,凝胶可以包裹在saran包装中并在4℃下储存
  2. 准备5x结合缓冲液。
  3. 设置绑定反应:
    1μlpoly-dIdC(TE中为1μg/μl)
    2μl5x结合缓冲液
    1μl标记的探针
    1μl冷竞争剂 - 含有结合序列的未标记DNA片段(如果需要)
    0.1μl100x BSA
    Xμl核提取物(总共5μg蛋白)
    将H sub 2 O加到10μl终体积中。
    在室温(RT)下孵育30分钟。 添加抗体(如果需要)。 在室温下再孵育30分钟。
  4. 当结合反应温育时,使用0.5×TBE作为运行缓冲液,在150V,30分钟时运行不含任何样品的聚丙烯酰胺凝胶。 然后在聚丙烯酰胺凝胶上在150V下运行样品〜2小时
  5. 干燥凝胶(可选)。
    转移凝胶到Whatman纸。 用纱布覆盖凝胶的顶部并在80℃下在真空干燥器中干燥1-2小时
  6. 暴露凝胶。
    将凝胶放在带反射屏幕的盒子中。 加入胶片并置于-80°C冰箱中。

食谱

  1. 5x结合缓冲液
    组合
    配方10ml,
    50mM Tris HCl(pH8.0) 0.5ml 1M Tris HCl(pH8.0)
    750 mM KCl
    3ml 2.5M KCl
    2.5mM EDTA
    50μl0.5M EDTA(pH8.0)
    0.5%Triton-X 100
    50微升Triton-X 100
    62.5%甘油(v/v) 7.87g甘油
    1 mM DTT
    使用前添加DTT
  2. 10x TBE缓冲液(1 L)
    106克Tris碱
    55克硼酸 40ml 0.5M EDTA(pH8.0)

致谢

这项工作由杭州高新区5050项目,杭州市海外退学基金资助,浙江省ZJ1000项目资助。 该协议是在美国加利福尼亚州斯坦福大学遗传学系Cohen实验室开发的。

参考文献

  1. Chen,R.,Liliental,J.E.,Kowalski,P.E.,Lu,Q.and Cohen,S.N。(2011)。 缺氧诱导因子-1alpha(HIF-1alpha)通过热休克因子HSF2和 HSF4。 Oncogene 30(22):2570-2580。
  • English
  • 中文翻译
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Copyright: © 2011 The Authors; exclusive licensee Bio-protocol LLC.
引用:Chen, R. (2011). A General EMSA (Gel-shift) Protocol. Bio-protocol Bio101: e24. DOI: 10.21769/BioProtoc.24;
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hayden thain
flinders uni
Hi I wanted to ask if its possible to use gel retardation on Sigma B as i believe it is the regulator of my target gene in S.aureus, or if there is a better protocol or method?

Thanks
1/8/2013 2:39:16 PM Reply
RAN CHEN
Stanford

Gel retardation is a good option if you know the candidate binding sites in the promoter region of your target gene. In your case, my opinion is that Ch-IP could be better choice.

1/10/2013 11:09:17 PM


Lin Fang
Department of Pediatrics, School of Medicine, Stanford University, USA
Hello, this is Lin Fang, associated editor of bio-protocol.org. The following is a organized answer to your question of our editorial board and the author Ran Chen.

Reporter assay in cells and Electric Mobility Shift Assay (EMSA) are often utilized to address the interaction between transcription factors and promoters. Reporter assay in cells is normally done first as it is often easier, quicker and more reliable in terms of whether transcription factors are involved in regulating promoter activities. EMSA can provide more details such as whether two transcription factors are present in two or the same complex. However, it is more tricky and prone to false results and requires high resolution, so often done after the reporter assays in cells. If interaction between A and B want to be assayed directly, Coimmunoprecipitation, GST-Pulldown and FRET (http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2937893/?tool=pubmed) can be performed.

Dual Luciferase Reporter Assays (Protocol is coming soon! )

Transfect cells respectively with the following combinations I-IV:
? I: pRL-CMV+ pGL3-G
? II: pRL-CMV+ pGL3-G + pA
? III: pRL-CMV + pGL3-G + pB
? IV: pRL-CMV + pGL3-G + pA + pB

? pRL-CMV: Promega E2261. pGL3: Promega E1751. pGL3-p: pGL3 reporter construct driven by promoter G. pA and pB: Transcription factor A and B in your favorite expression vector.
? Control groups I’-IV’ should also be included except that pGL3-G in corresponding group A-D is replaced by pGL3 which is a promoterless reporter constructs.

Analyze the transfection with Dual Luciferase Assay (Promega E1910). Expected results are
? Here, pGL3-G should be much stronger than pGL3 (5 fold) to confirm true activation.
? If addition of A and/or B change the promoter activity of G, A and/or B bind to promoter G.
? If addition of both A and B show additive effect of addition of A and B alone, it suggests that A and B MAY NOT bind each other.
? If addition of both A and B show synergistic effect of addition of A and B alone, it suggests that A and B MAY bind each other.
To further delineate the binding sites of A and B on promoter G, potential binding sites of A and B can be predicted according to their canonical binding sequences and mutated. The effect of mutations in promoter G can be assayed with the reporter assay.
If high activation pGL3-G in the absence of A or B, siRNA of A or B can be tested too.

EMSA in the presence of competing DNA sequences and antibodies against A and B respectively
Before proceeding, it is desirable to narrow the labeled DNA sequences as well as non-labeled competing DNA sequences to shorter length. So it is better to narrow down the sequence by footprinting and identification of potential binding sites of A and B referring to canonical A and B binding sites.
Material:
? Cell nuclear extract (make sure it contains A and B)
? Labeled DNA sequence
? Unlabeled DNA sequence
? A antibody
? B antibody
? Non-immune serum
Native-PAGE gel
Mix in the following combinations
1. Labeled DNA
2. Labeled DNA + Cell nuclear extract
3. Labeled DNA + Cell nuclear extract + unlabeled DNA
4. Labeled DNA + Cell nuclear extract + A antibody
5. Labeled DNA + Cell nuclear extract + B antibody
6. Labeled DNA + Cell nuclear extract + non-immune serum
Run the reaction in native PAGE gel

Expected results:
? Labeled DNA + Cell nuclear extract should form several bands, containing specific and nonspecific complex.
? Bands representing specific complex should disappear in the presence of unlabeled DNA.
? If transcription factor A or B is present in the complexes, their corresponding antibody should supershif the specific complex, aka, running slower in the PAGE gel.
? If A and B antibody supershift the same band, A and B form one complex.
? Non-immune serum should not supershift the specific complex.






1/24/2012 5:58:09 AM Reply
Please design a protocal to identify whether transcriptional factor A and transcriptional factor B may bind together to form a complex, and bind to the promoter of G gene to increase its transcriptional level. (Please use the folowing techniqes: Yeast two-hibrid, ChIP, EMSA, Reporter gene assay)
1/17/2012 2:16:15 PM Reply