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Analysis of RNA-protein Interactions Using Electrophoretic Mobility Shift Assay (Gel Shift Assay)
EMSA实验(凝胶迁移试验)分析RNA-蛋白质的相互作用

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Abstract

RNA binding proteins (RBPs) play a crucial role in regulating gene expression at the post-transcriptional level at multiple steps including pre-mRNA splicing, polyadenylation, mRNA stability, mRNA localization and translation. RBPs regulate these processes primarily by binding to specific sequence elements in nascent or mature transcripts. There are several hundreds of RBPs in plants, but the targets of most of them are unknown. A variety of experimental methods have been developed to identify targets of an RBP. These include RNA immunoprecipitation (RIP), UV cross-linking and immunoprecipitation (CLIP) and many variations of CLIP (e.g. PAR-CLIP, iCLIP). These approaches depend on immunoprecipitation of RNAs bound to a specific RBP using an antibody to that RBP. Electrophoretic mobility shift assay (EMSA), also called gel shift assay, has been used to analyze protein-nucleic acid interactions. It is a simple and powerful method to analyze protein-RNA/DNA interactions. In RNA EMSA, RNA-protein complexes are visualized by comparing the migration of RNA in the presence of a protein. Generally, in RNA EMSA a specific RNA sequence is used to analyze its interaction with a protein. In vitro transcribed 32P labeled or chemically synthesized RNA with a fluorescent tag is incubated with or without the protein of interest and the reaction mixture is then run on native polyacrylamide gel electrophoresis. RNA-Protein complexes migrate slowly as compared to free RNA, which can be visualized using an imaging system. In addition to test binding of an RBP to RNA, EMSA is also used to map the region in RNA and/or protein that is involved in interaction. Furthermore, the binding affinity can also be quantified using EMSA.

Materials and Reagents

Note: All work should be done in an RNase free environment, using only sterile, RNase free solutions and materials.

  1. E. coli BL21 (DE3)-pLysS host cells harboring SR45 cDNA
  2. In vitro transcribed RNA
  3. Purified recombinant protein
  4. Cap analogue (New England Biolabs, catalog number: S1404S )
  5. SP6 polymerase (Fermentas, catalog number: EP0131 20 U/μl)
  6. rNTPs
    ATP (Fermentas, catalog number: R0441 )
    CTP (Fermentas, catalog number: R0451 )
    GTP (Fermentas, catalog number: R0461 )
    UTP (Fermentas, catalog number: R0471 )
  7. RNase Inhibitor (Life Technologies, Invitrogen™, catalog number: 10777019 )
  8. 32P-UTP- Uridine 5’ triphosphate (PerkinElmer, catalog number: BLU007C001 )
  9. HEPES (Thermo Fischer Scientific, catalog number: AC172571000 )
  10. KCl (Sigma-Aldrich, catalog number: P9541 )
  11. MgCl2 (Mallinckrodt, catalog number: 5958-04 )
  12. Glycerol (Thermo Fischer Scientific, catalog number: G331 )
  13. Triton X-100 (Thermo Fischer Scientific, catalog number: BP151 )
  14. Sodium dodecyl sulfate (SDS) (Mallinckrodt Baker, catalog number: 4095-02 )
  15. NaCl (Thermo Fischer Scientific, catalog number: 7647-14-5 )
  16. Na2HPO4 (Mallinckrodt, catalog number: 7917-04 )
  17. KH2PO4 (Thermo Fischer Scientific, catalog number: 7778-77-0 )
  18. Spermidine (Sigma-Aldrich, catalog number: 52626 )
  19. Heparin sulfate (Sigma-Aldrich, catalog number: H4784 )
  20. Tris base (AMRESCO, catalog number: 77-86-1 )
  21. Acrylamide/bis-acrylamide (Bio-Rad Laboratories, catalog number: 16105B )
  22. Phenol: chloroform (AMRESCO, catalog number: 0883 )
  23. Ammonium persulfate (APS) (Gibco BRL®, catalog number: 5523UA )
  24. TEMED (VWR International, catalog number: 97064-684 )
  25. Bromophenol blue (Sigma-Aldrich, catalog number: 161-0404 )
  26. Xylene cyanol (Sigma-Aldrich, catalog number: X4126 )
  27. pGEM vector (Promega, catalog number: P2161 )
  28. Isopropyl-β-D thiogalactopyranoside (IPTG) (Gold Bio, catalog number: I2481C5 )
  29. Ampicillin (VWR International, catalog number: IB02040 )
  30. S-Protein agarose beads (Merck KGaA, Novagen®, catalog number: 80031-014 )
  31. Lysozyme (Sigma-Aldrich, catalog number: L6876 )
  32. pET32c vector (Merck KGaA, Novagen®, catalog number: 69017-3 )
  33. Protease inhibitor cocktail (Sigma-Aldrich, catalog number: P9599 )
  34. Boric acid (Mallinckrodt, catalog number: 2549-04 )
  35. Ethylenediaminetetraacetic acid (EDTA) (Thermo Fischer Scientific, catalog number: S311-500 )
  36. Ammonium acetate (Thermo Fischer Scientific, catalog number: A637-500 )
  37. Bovine serum albumin (BSA)
  38. LB medium
  39. 5x Gel shift buffer (see Recipes)
  40. Lysis buffer (see Recipes)
  41. 10x TBE (see Recipes)
  42. Gel Mix (see Recipes)
  43. 5% Denature Gel (see Recipes)
  44. 5% Non-Denaturing Gel (see Recipes)
  45. TNS Solution (see Recipes)
  46. RNA loading dye for Urea gel (see Recipes)
  47. 6x loading buffer for Non-Urea gel (see Recipes)
  48. Binding/wash buffers (see Recipes)
  49. Phosphate buffer (see Recipes)
  50. Citrate buffer (see Recipes)

Equipment

  1. 1.5 ml microcentrifuge tube (BioExpress, catalog number: C-3262 )
  2. Saran wrap
  3. Whatman filter paper
  4. Water bath (VWR International, model: 1225 PC )
  5. Electrophoresis equipment with power supply (Thermo Fisher Scientific, model: B2 easy cost )
  6. Gel drying apparatus (Savant System LLC, model: SGD 2000 )
  7. Phosphorimager (Molecular Dynamics, model: Storm 840 )
  8. Phosphorimager screen (Molecular Dynamics, model: 1000004864 )
  9. Sonicator (SP Scientific, model: 274480 )
  10. 0.45 micron syringe filter (Life Science Products, catalog number: 25CSO80AS )
  11. Centrifuge (Eppendorf, model: 022620401 )
  12. Vertical gel apparatus (Gibco BRL®, model: V15.17 )
  13. Liquid scintillation counter
  14. Orbital shaker

Procedure

Note: We routinely produce RNA probes by in vitro transcription using a linearized pGEM clone that contains the DNA corresponding to RNA of interest. pGEM vector has T7 and SP6 promoters. Depending on the orientation of insert DNA either T7 polymerase or SP6 polymerase is used in in vitro transcription to generate RNA probe.

  1. Preparation of RNA using in vitro transcription system
    1. Prepare the following reaction in a 1.5 ml of microcentrifuge tube. 1 μl DNA template (~ 1 μg of linearized plasmid DNA), 1 μl 10x RNA polymerase buffer, 1 μl rNTPs (5 mM ATP, CTP, 0.5 mM GTP, UTP), 1 μl cap analog (5 mM), 4.5 μl (45 μCi): a-32P-UTP (800 Ci/mmol; 10 mCi/ml), 0.5 μl RNase Inhibitor, and 1 μl SP6 polymerase a total of 10 μl of reaction.
    2. Incubate 37 °C, for 3 to 6 hours (Note 1).
    3. Add 90 μl dH2O to 10 μl total 100 μl.
    4. Add equal volume of (100 μl) of phenol/chloroform, mix by vortexing.
    5. Spin at 16,000 x g for 5 min.
    6. Transfer upper layer into a new Eppendorf (~100 μl).
    7. Add 33 μl 10 M ammonium acetate and 250 μl 100% ethanol to the 100 μl.
    8. Incubate at -80 °C for 10 min.
    9. Spin at 16,000 x g for 10 min.
    10. Wash the pellet with 500 μl of 80% ethanol.
    11. Air dry the pellet; resuspend in 10 μl RNA loading dye.
    12. Heat 1 min at 90 °C.
    13. Load on 5% denaturing acrylamide gel and run for one to two hours depending on the size of RNA (Note 2).
    14. Disassemble the gel chamber and dismantle the gel, leaving it mounted on one plate. To facilitate the alignment of the gel to the X-ray film in stepA16 below, cut one corner of the gel.
    15. Wrap the gel with Saran wrap to avoid contamination.
    16. Expose the gel to an X-ray film for 2-5 min and develop the film to visualize radioactive bands.
    17. Align the gel on top of the X-ray film and locate the region in the gel corresponding to the radioactive band on the film.
    18. Excise the gel corresponding to the band on the X-ray film (see Figure 1 and Notes 3 & 4).


      Figure 1. In vitro transcribed RNA used in Figure 2a and 2b. After exposing the gel to an X-ray film, identified the right size of RNA product, excised the gel corresponding to the band and used to extract RNA.

    19. Transfer the excised band to 400 μl of TNS solution in an Eppendorf tube and incubate overnight at room temperature.
    20. Next day, take out TNS solution, which contains radiolabelled RNA and add equal volume of phenol/chloroform, mix by vortexing.
    21. Spin at 16,000 x g for 10 min.
    22. Wash the pellet with 500 μl of 80% ethanol.
    23. Dry the pellet; resuspend the RNA pellet in 20 μl H2O.
    24. Measure radioactivity in1 μl of RNA using liquid scintillation counter and use this number to calculate concentration of labeled “U” residues in RNA probe (Note 5).
    25. Dilute RNA probe with RNase free water to 50 to100 K cpm/μl for use in EMSA assays.

  2. Preparation and purification of recombinant protein
    For EMSA analysis apart from RNA a purified protein of interest is needed. One can generate this protein by cloning the cDNA into a bacterial expression vector. Here we expressed SR45 in E.coli as an S.tag fusion and purified it using S-protein agarose beads.
    1. Grow E. coli BL21 (DE3)-pLysS host cells harboring SR45 cDNA in LB medium containing ampicillin at 37 °C for overnight. Next day inoculate 1 ml of overnight culture into 100 ml of LB medium containing appropriate antibiotic, then grow culture until OD600 reaches ~0.6 (it will take approximately 2 h).
    2. Induce expression of recombinant protein by adding IPTG to a final concentration of 0.5 mM and allow the bacterial culture to grow for an additional 4 h at 30 °C.
    3. Harvest bacterial cells by centrifugation at 2,350 x g for 10 min at 4 °C.
    4. Discard the supernatant and resuspend the cell pellet in 1/10 culture volume (5 ml) of 50 mM Tris-HCl pH 8.0, 2 mM EDTA.
    5. Add lysozyme to a concentration of 100 μg/ml; use a 10 mg/ml stock freshly prepared in 50 mM Tris-HCl pH 8.0, 2 mM EDTA. Then add 1/10 volume (0.5 ml) of 1% Triton X-100. Incubate at 30 °C for 15 min.
    6. Place the tube in an ice bath and sonicate 4 times (at 3.5 setting) for 15 sec/each time.
    7. Centrifuge at 12,000 x g for 15 min at 4 °C. The supernatant contains soluble proteins.
    8. Filter the supernatant through a 0.45 micron membrane.
    9. Add 100 μl of binding/wash buffer to 100 μl of S-protein Agarose slurry in an Eppendorf tube and mix it gently, then add 1 ml of soluble proteins from step 8 (Note 6).
    10. Mix and incubate at room temperature on an orbital shaker for 30 min. (Do not shake vigorously as this may denature protein).
    11. Centrifuge at 500 x g for 10 min at 4 °C and carefully decant supernatant and wash the beads with five times, 1 ml each time, with binding/wash buffer.
    12. Resuspend the washed beads containing the bound protein in 150 μl of the 0.2 M citrate buffer, pH 2.
    13. Incubate for 10 min at room temperature, mix gently every few minutes.
    14. Neutralized by adding 8 μl 2 M Tris-base (pH 10.4).
    15. The eluted proteins were dialyzed against phosphate buffer (Note 7).

  3. Electrophoretic Mobility Shift Assay (EMSA)
    Before setting up the reaction, prepare 5% non-denaturing (native) gel, and pre-run the gel at 200 V using 1x TBE buffer for 10 to 15 minutes.
    1. Prepare the following reaction mixture (final volume 14 μl) in a 1.5 ml of microcentrifuge tube. 1.5 μl of 1 mM spermidine (100 mM), 3 μl of 5x gel shift buffer, 0.5 μl of RNase Inhibitor, 1 μl of radiolabel RNA (50-100 K cpm), x μl lysis buffer (depending on protein volume) and x μl of protein of interest (Note 8).
    2. Incubate at 30 °C for 5 min.
    3. Add 1 μl of Heparin Sulfate (50 mg/ml) (Note 9).
    4. Transfer the reactions to ice for 5 min.
    5. Add 3 μl 6x urea free loading buffer.
    6. Load the reaction products on 5% non-denaturing gel and run at 200 V for 2 to 3 h or as long as necessary for good separation (normally we will run 2 h for RNA probe that is about 200 nt) (Note 10).
    7. Transfer the gel to Whatman filter paper, cover with Saran wrap, and dry with a gel-drying apparatus.
    8. The dried gel should be exposed to the phosphorimaging screen for 2 h to overnight.
    9. Free RNA and RNA – Protein complex(es) are visualized by using phosphorimager (see Figure 2a and 2b).


      Figure 2. RNA EMSA. A. EMSA with an RNA probe using a purified recombinant protein. Lane 1, free probe; lanes 2-5, increasing concentration of recombinant protein. B. Binding of recombinant protein to RNA probe is competed by cold RNA. Lane 1, free probe; lane 2, probe + recombinant protein; lanes 3-7, as lane 2, with increasing concentration of cold RNA. Arrows indicate free probe, and the RNA-protein complex is indicated by arrowheads.

  4. Competition with unlabeled RNA
    After confirming the binding of a protein with a specific RNA target using EMSA, the specificity of interaction can be tested by competition studies in the presence of excess amount of same RNA that is not labeled. Increasing amount of competitor RNA (up to 100x of labeled RNA) is added to the binding reaction mixture and the ability of the cold competitor RNA to disrupt the complex is determined as above using native gel electrophoresis (see Figure 2b). Unlabeled competitor RNAs is generated in the same manner as above (see A section), except that the amount of cold UTP is increased to 5 mM and the amount of radioactive UTP is reduced to 0.045 μCi.

Notes

  1. Three hours of incubation yields good amount of RNA.
  2. For ~100 base long RNA probe run the gel for 1 h; for 200 to 300 nts long probe run the gel for two to three hours.
  3. It is important to run in vitro transcribed RNA on a denaturing gel to confirm that the right size product is generated. The correct size band should then be excised and purified.
  4. Sometimes RNA templates yield two or more RNA products due to its secondary structure. If this happens, excise only the correct size band for purification.
  5. Concentration of labeled “U” in RNA is calculated using the specific activity of radiolabeled UTP, final concentration of UTP (radiolabeled UTP plus cold UTP) in the reaction and the number of “U” residues in the template. (fmoles of RNA = fmoles of UTP in the RNA probe/number of "U" residues in the labeled RNA).
  6. Since the amount of protein in soluble fraction varies depending on the clone and expression level, it is necessary to optimize the ratio between agarose beads and the protein.
  7. The recombinant protein should be purified to near homogeneity and protein concentration should be determined. It is not advisable to use crude extract from bacteria expressing recombinant protein.
  8. Always use a known concentration of RNA probe and increasing concentration of protein.
  9. Heparin sulfate reduces non-specific binding of RNA probe to proteins and eliminates background.
  10. Running time of native gels is dependent on the RNA and protein complex, it must be optimized for each RNA and protein. For longer run times, gels must be run at 4 °C temperature.
  11. Smaller size RNA probes (from 50 to 200 nts) work well in RNA EMSA.
  12. If some RNA/protein complex stays in the well, add BSA to minimize this effect.
  13. The concentration of RNA and protein should be optimized for each RNA-protein complex. Approximately 100,000 cpm of RNA and 500 ng of protein is a good starting point for binding studies.

Recipes

   Note: All buffers should be prepared with RNase free water.

  1. 5x Gel shift buffer
    70 mM HEPES pH 7.9
    450 mM KCl
    11 mM MgCl2
    28% Glycerol
  2. Lysis buffer
    50 mM HEPES pH 7.9
    150 mM KCl
    1 mM MgCl2
    1% Triton X–100
    10% Glycerol
  3. 10x TBE
    108 grams of Tris base
    55 grams of boric acid
    9.3 grams of EDTA dissolved in water
    Made up to one liter and autoclaved
    Use 1x as running buffer in running both denaturing and non-denaturing (Native) gels
  4. Gel Mix (500 ml)
    240 grams of Urea
    50 ml of 10x TBE
    200 ml H2O
    Gel mix is stable for at least two to three months at room temperature
  5. 5% Denature Gel (30 ml)
    5 ml of 30% Acrylamide/bisacrylamide
    25 ml gel mix
    300 μl 10% APS
    30 μl TEMED
  6. 5% Non-Denaturing Gel
    6.75 ml of 40% Acrylamide/bisacrylamide (38:2)
    4.5 ml of 10x TBE
    300 μl of 10% APS
    30 μl of TEMED
    33.6 ml of H2O
  7. TNS Solution
    25 mM Tris-HCl pH 7.5
    400 mM NaCl
    0.1% SDS
  8. RNA loading dye for Urea gel
    20 mM Tris-HCl pH 7.6
    8 M Urea
    1 mM EDTA
    0.05% Xylene cyanol
    0.05% Bromophenolo blue
  9. 6x loading buffer for Non-Urea gel
    30% Glycerol
    0.3% Bromophenol Blue
    0.3% Xylene Cyanol
  10. Binding/wash buffers
    20 mM Tris-HCl, pH 7.5
    150 mM NaCl
    0.1% Triton X-100
    1x protease inhibitors
  11. Phosphate buffer
    10 mM Na2HPO4
    2 mM KH2PO4
    2.7 mM KCl
    137 NaCl pH 7.4
  12. Citrate Buffer
    2 M citric acid
    Adjust pH to 2 with 10 M KOH and dilute to 0.2 M

Acknowledgments

This protocol was adapted from Thomas et al. (2012). This work was supported by a grant from the US National Science Foundation.

References

  1. Day, I. S., Golovkin, M., Palusa, S. G., Link, A., Ali, G. S., Thomas, J., Richardson, D. N. and Reddy, A. S. (2012). Interactions of SR45, an SR-like protein, with spliceosomal proteins and an intronic sequence: insights into regulated splicing. Plant J 71(6): 936-947.
  2. Golovkin, M. and Reddy, A. S. (1999). An SC35-like protein and a novel serine/arginine-rich protein interact with Arabidopsis U1-70K protein. J Biol Chem 274(51): 36428-36438.
  3. Palusa, S. G. and Wilusz, J. (2013). Approaches for the Identification and Characterization of RNA-Protein Interactions. Biophysical approaches to translational control of gene expression, Biophysics for the Life Sciences, J. D. Dinman (eds). Springer: 199-212.
  4. Ryder, S. P., Recht, M. I. and Williamson, J. R. (2008). Quantitative analysis of protein-RNA interactions by gel mobility shift. Methods Mol Biol 488: 99-115. 
  5. Thomas, J., Palusa, S. G., Prasad, K. V., Ali, G. S., Surabhi, G. K., Ben-Hur, A., Abdel-Ghany, S. E. and Reddy, A. S. (2012). Identification of an intronic splicing regulatory element involved in auto-regulation of alternative splicing of SCL33 pre-mRNA. Plant J 72(6):935-946. 
  6. Wilusz, J. and Shenk, T. (1988). A 64 kd nuclear protein binds to RNA segments that include the AAUAAA polyadenylation motif. Cell 52(2): 221-228.

简介

RNA结合蛋白(RBP)在包括前mRNA剪接,多聚腺苷酸化,mRNA稳定性,mRNA定位和翻译的多个步骤在调节转录后水平的基因表达中起关键作用。 RBP主要通过结合新生或成熟转录物中的特定序列元件来调节这些过程。植物中有几百个RBP,但其中大多数的目标是未知的。已经开发了各种实验方法来鉴定RBP的目标。这些包括RNA免疫沉淀(RIP),UV交联和免疫沉淀(CLIP)和CLIP的许多变体(例如PAR-CLIP,iCLIP)。这些方法取决于使用针对该RBP的抗体与特异性RBP结合的RNA的免疫沉淀。电泳迁移率变动分析(EMSA),也称为凝胶移位分析,已被用于分析蛋白质 - 核酸相互作用。它是一种简单而强大的方法来分析蛋白质-RNA/DNA相互作用。在RNA EMSA中,通过在蛋白质存在下比较RNA的迁移来可视化RNA-蛋白质复合物。通常,在RNA EMSA中,使用特异性RNA序列来分析其与蛋白质的相互作用。将具有荧光标记的体外转录的32 P标记的或化学合成的RNA与或不与目标蛋白一起温育,然后将反应混合物在天然聚丙烯酰胺凝胶电泳上运行。 RNA-蛋白复合物与游离RNA相比缓慢迁移,其可以使用成像系统可视化。除了测试RBP与RNA的结合之外,EMSA还用于绘制参与相互作用的RNA和/或蛋白质中的区域。此外,还可以使用EMSA定量结合亲和力。

材料和试剂

注意:所有的工作都应该在无RNA酶的环境中进行,只使用无菌,无RNase的解决方案和材料。

  1. E。 含有SR45 cDNA的大肠杆菌 BL21(DE3)-pLysS宿主细胞
  2. 体外转录的RNA
  3. 纯化的重组蛋白
  4. Cap类似物(New England Biolabs,目录号:S1404S)
  5. SP6聚合酶(Fermentas,目录号:EP013120U /μl)
  6. rNTPs
    ATP(Fermentas,目录号:R0441)
    CTP(Fermentas,目录号:R0451)
    GTP(Fermentas,目录号:R0461)
    UTP(Fermentas,目录号:R0471)
  7. RNase抑制剂(Life Technologies,Invitrogen TM,目录号:10777019)
  8. 32 P-UTP-尿苷5'三磷酸(PerkinElmer,目录号:BLU007C001)
  9. HEPES(Thermo Fischer Scientific,目录号:AC172571000)
  10. KCl(Sigma-Aldrich,目录号:P9541)
  11. MgCl 2(Mallinckrodt,目录号:5958-04)
  12. 甘油(Thermo Fischer Scientific,目录号:G331)
  13. Triton X-100(Thermo Fischer Scientific,目录号:BP151)
  14. 十二烷基硫酸钠(SDS)(Mallinckrodt Baker,目录号:4095-02)
  15. NaCl(Thermo Fischer Scientific,目录号:7647-14-5)

  16. (Mallinckrodt,目录号:7917-04)。

  17. (Thermo Fischer Scientific,目录号:7778-77-0)
  18. 亚精胺(Sigma-Aldrich,目录号:52626)
  19. 硫酸肝素(Sigma-Aldrich,目录号:H4784)
  20. Tris碱(AMRESCO,目录号:77-86-1)
  21. 丙烯酰胺/双丙烯酰胺(Bio-Rad Laboratories,目录号:16105B)
  22. 苯酚:氯仿(AMRESCO,目录号:0883)
  23. 过硫酸铵(APS)(Gibco BRL ,目录号:5523UA)
  24. TEMED(VWR International,目录号:97064-684)
  25. 溴酚蓝(Sigma-Aldrich,目录号:161-0404)
  26. 二甲苯Cyanol(Sigma-Aldrich,目录号:X4126)
  27. pGEM载体(Promega,目录号:P2161)
  28. 异丙基-β-D硫代吡喃半乳糖苷(IPTG)(Gold Bio,目录号:I2481C5)
  29. 氨苄青霉素(VWR International,目录号:IB02040)
  30. S-蛋白琼脂糖珠(Merck KGaA,Novagen ,目录号:80031-014)
  31. 溶菌酶(Sigma-Aldrich,目录号:L6876)
  32. pET32c载体(Merck KGaA,Novagen ,目录号:69017-3)
  33. 蛋白酶抑制剂混合物(Sigma-Aldrich,目录号:P9599)
  34. 硼酸(Mallinckrodt,目录号:2549-04)
  35. 乙二胺四乙酸(EDTA)(Thermo Fischer Scientific,目录号:S311-500)
  36. 乙酸铵(Thermo Fischer Scientific,目录号:A637-500)
  37. 牛血清白蛋白(BSA)
  38. LB培养基
  39. 5x凝胶移位缓冲液(参见配方)
  40. 裂解缓冲液(见配方)
  41. 10x TBE(参见配方)
  42. 凝胶混合(见配方)
  43. 5%变性凝胶(见配方)
  44. 5%无变性凝胶(见配方)
  45. TNS解决方案(参见配方)
  46. 尿素凝胶的RNA加载染料(参见配方)
  47. 用于非尿素凝胶的6x装载缓冲液(参见配方)
  48. 结合/洗涤缓冲液(参见配方)
  49. 磷酸盐缓冲液(参见配方)
  50. 柠檬酸缓冲液(见配方)

设备

  1. 1.5ml微量离心管(BioExpress,目录号:C-3262)
  2. Saran换装
  3. Whatman过滤纸
  4. 水浴(VWR International,型号:1225 PC)
  5. 具有电源的电泳设备(Thermo Fisher Scientific,型号:B2容易成本)
  6. 凝胶干燥设备(Savant System LLC,型号:SGD 2000)
  7. Phosphorimager(Molecular Dynamics,型号:Storm 840)
  8. 荧光屏(Molecular Dynamics,型号:1000004864)
  9. Sonicator(SP Scientific,型号:274480)
  10. 0.45微米注射器过滤器(Life Science Products,目录号:25CSO80AS)
  11. 离心机(Eppendorf,型号:022620401)
  12. 垂直凝胶装置(Gibco BRL ,型号:V15.17)
  13. 液体闪烁计数器
  14. 轨道振动器

程序

注意:我们常规地通过使用含有对应于感兴趣的RNA的DNA的线性化pGEM克隆体外转录产生RNA探针。 pGEM载体具有T7和SP6启动子。 取决于插入DNA的取向,T7聚合酶或SP6聚合酶用于体外转录以产生RNA探针。

  1. 使用体外转录系统制备RNA
    1. 在1.5 ml微量离心管中制备以下反应。 将1μlDNA模板(约1μg线性化的质粒DNA),1μl10x RNA聚合酶缓冲液,1μlrNTP(5mM ATP,CTP,0.5mM GTP,UTP),1μl盖帽类似物(5mM) 45μCi):a- 32 P-UTP(800Ci/mmol; 10mCi/ml),0.5μlRNA酶抑制剂和1μlSP6聚合酶,总共10μl反应物。 >
    2. 孵育37℃,3至6小时(注1)。
    3. 加入90μldH 2 O至10μl总计100μl
    4. 加入等体积(100μl)的苯酚/氯仿,涡旋混合
    5. 旋转16,000英寸x g 5分钟。
    6. 将上层转移到一个新的Eppendorf(〜100微升)
    7. 加入33μl10M乙酸铵和250μl100%乙醇到100μl
    8. 在-80℃孵育10分钟。
    9. 旋转16,000英寸x g 10分钟。
    10. 用500μl80%乙醇洗涤沉淀。
    11. 空气干燥颗粒; 重悬于10μlRNA加载染料中
    12. 在90℃下加热1分钟
    13. 加载在5%变性丙烯酰胺凝胶上,运行一到两个小时,取决于RNA的大小(注2)。
    14. 拆卸凝胶室并拆除凝胶,将其安装在一块板上。 为了便于在下面的步骤A16中凝胶与X射线胶片的对准,切割凝胶的一个角
    15. 用Saran包装包裹凝胶以避免污染。
    16. 将凝胶暴露于X射线胶片2-5分钟,并显影该胶片以显现放射性带
    17. 将凝胶对准X射线胶片的顶部,并找到凝胶上对应于胶片上放射性带的区域。
    18. 切除相应于X射线胶片上的条带的凝胶(见图1和注释3和4)。


      图1. 在图2a和2b中使用的体外转录的RNA 。将凝胶暴露于X射线胶片后,鉴定适当大小的RNA产物,切下凝胶对应于条带并用于提取RNA
    19. 将切除的带转移到400微升TNS溶液在Eppendorf管中,并在室温下孵育过夜。
    20. 第二天,取出TNS溶液,其中含有放射性标记的RNA,加入等体积的苯酚/氯仿,涡旋混合。
    21. 旋转16,000英寸x g 10分钟。
    22. 用500μl80%乙醇洗涤沉淀。
    23. 干燥沉淀; 将RNA沉淀重悬于20μlH 2 O中
    24. 使用液体闪烁计数器测量1μlRNA中的放射性,并使用此数值计算RNA探针中标记的"U"残基的浓度(注5)。
    25. 用RNA酶自由水稀释RNA探针至50至100 K cpm /μl,用于EMSA检测
  2. 重组蛋白的制备和纯化
    对于除RNA之外的EMSA分析,需要纯化的目的蛋白。可以通过将cDNA克隆到细菌表达载体中来产生这种蛋白质。这里,我们在大肠杆菌中表达 SR45 作为S.tag融合体,并使用S-蛋白琼脂糖珠纯化。
    1. 成长。在含有氨苄青霉素的LB培养基中将含有SR45 cDNA的大肠杆菌BL21(DE3)-pLysS宿主细胞在37℃下过夜。第二天将1ml过夜培养物接种到含有适当抗生素的100ml LB培养基中,然后培养直至OD 600达到〜0.6(大约需要2小时)。
    2. 通过加入IPTG至终浓度为0.5mM诱导重组蛋白的表达,并使细菌培养物在30℃下再生长4小时。
    3. 通过在4℃下以2,350×g离心10分钟收获细菌细胞
    4. 弃去上清液,并在50mM Tris-HCl pH 8.0,2mM EDTA的1/10培养物体积(5ml)中重悬细胞沉淀。
    5. 加入溶菌酶至100μg/ml的浓度; 使用在50mM Tris-HCl pH 8.0,2mM EDTA中新鲜制备的10mg/ml储备液。 然后加入1/10体积(0.5ml)的1%Triton X-100。 在30℃孵育15分钟。
    6. 将管放置在冰浴中,超声4次(3.5设置),每次15秒。
    7. 在4℃下以12,000xg离心15分钟。 上清液含有可溶性蛋白质
    8. 通过0.45微米的膜过滤上清液
    9. 在Eppendorf管中加入100μl结合/洗涤缓冲液到100μlS蛋白琼脂糖浆中,轻轻混合,然后加入1 ml步骤8中的可溶性蛋白(注释6)。
    10. 混合并在定轨振荡器上室温孵育30分钟。 (不要剧烈摇动,因为这可能会使蛋白质变性)。
    11. 在4℃下以500×g离心10分钟,小心地倾析上清液,并用结合/洗涤缓冲液洗涤珠粒,每次5次,每次1ml。
    12. 将含有结合蛋白的洗过的珠子重悬于150μlpH2的0.2M柠檬酸缓冲液中
    13. 在室温下孵育10分钟,每隔几分钟轻轻混匀
    14. 通过加入8μl2M Tris-碱(pH 10.4)中和。
    15. 洗脱的蛋白质对磷酸盐缓冲液(注7)透析
  3. 电泳迁移率变动分析(EMSA)
    在设置反应前,制备5%非变性(天然)凝胶,并使用1×TBE缓冲液在200V预运行凝胶10至15分钟。
    1. 在1.5ml微量离心管中制备以下反应混合物(终体积14μl)。 1.5μl1mM亚精胺(100mM),3μl5×凝胶移位缓冲液,0.5μlRNA酶抑制剂,1μl放射性标记RNA(50-100Kcpm),xμl裂解缓冲液(取决于蛋白质体积)和x μl的目的蛋白质(注8)
    2. 在30℃孵育5分钟。
    3. 加入1μl硫酸肝素(50mg/ml)(注9)
    4. 将反应物转移到冰上5分钟
    5. 加入3微升6倍尿素的加载缓冲液
    6. 将反应产物装载在5%非变性凝胶上,并在200V运行2至3小时或者为了良好分离(通常对于约200nt的RNA探针,将运行2小时)(注10)。
    7. 将凝胶转移到Whatman滤纸上,用Saran包裹物覆盖,并用凝胶干燥设备干燥
    8. 干燥的凝胶应该暴露于荧光成像屏2小时至过夜
    9. 游离RNA和RNA - 蛋白复合物通过使用phosphorimager显现(见图2a和2b)

      图2.RNA EMSA。 A。 EMSA与使用纯化的重组蛋白的RNA探针。泳道1,游离探针;泳道2-5,增加重组蛋白的浓度。 B.通过冷RNA竞争重组蛋白与RNA探针的结合。泳道1,游离探针;泳道2,探针+重组蛋白;泳道3-7,作为泳道2,具有渐增浓度的冷RNA。箭头表示游离探针,RNA-蛋白复合物用箭头表示
  4. 与未标记的RNA的竞争
    在使用EMSA证实蛋白质与特异性RNA靶标的结合之后,可以通过在未标记的过量的相同RNA存在下的竞争研究来测试相互作用的特异性。向结合反应混合物中加入增加量的竞争剂RNA(高达100x的标记的RNA),并且如上使用天然凝胶电泳测定冷竞争剂RNA破坏复合物的能力(参见图2b)。未标记的竞争RNA以与上述相同的方式产生(参见A部分),除了冷UTP的量增加至5mM,并且放射性UTP的量减少至0.045μCi。

笔记

  1. 孵育3小时产生大量的RNA
  2. 对于〜100碱基长的RNA探针运行凝胶1小时;对于200至300nt长的探针,运行凝胶2至3小时
  3. 重要的是在变性凝胶上运行体外转录的RNA以确认产生正确大小的产物。然后应该切除和纯化正确的大小带。
  4. 有时RNA模板由于其二级结构而产生两种或更多种RNA产物。如果发生这种情况,只有正确的尺寸带进行纯化。
  5. 使用放射性标记的UTP的比活性,UTP的最终浓度(放射性标记的UTP加冷UTP)在反应中的浓度和模板中"U"残基的数目来计算RNA中标记的"U"的浓度。 (RNA的摩尔数= RNA探针中UTP的f摩尔数/标记的RNA中"U"残基的数目)。
  6. 由于可溶性级分中的蛋白质量根据克隆和表达水平而变化,因此有必要优化琼脂糖珠粒和蛋白质之间的比例。
  7. 重组蛋白应纯化至接近均匀,并应测定蛋白浓度。不建议使用表达重组蛋白的细菌的粗提取物
  8. 始终使用已知浓度的RNA探针和增加蛋白质的浓度
  9. 硫酸肝素降低RNA探针与蛋白质的非特异性结合并消除背景
  10. 天然凝胶的运行时间取决于RNA和蛋白质复合物,必须针对每种RNA和蛋白质进行优化。 对于更长的运行时间,凝胶必须在4°C的温度下运行。
  11. 较小尺寸的RNA探针(50至200个核苷酸)在RNA EMSA中工作良好
  12. 如果一些RNA /蛋白复合物停留在孔中,则加入BSA以使这种影响最小化
  13. 应针对每个RNA-蛋白复合物优化RNA和蛋白质的浓度。 大约100,000 cpm的RNA和500 ng蛋白质是结合研究的良好起点

食谱

   注意:所有缓冲液应使用不含RNase的水制备。

  1. 5x凝胶移位缓冲液
    70 mM HEPES pH 7.9
    450 mM KCl
    11mM MgCl 2
    28%甘油
  2. 裂解缓冲液
    50 mM HEPES pH 7.9
    150 mM KCl
    1mM MgCl 2
    1%Triton X-100 10%甘油
  3. 10x TBE
    108克Tris碱
    55克硼酸
    9.3克溶于水中的EDTA 加至一升,高压灭菌
    使用1x作为运行缓冲液运行变性和非变性(本地)凝胶
  4. 凝胶混合物(500ml) 240克尿素
    50ml 10×TBE
    200ml H 2 O 2 / 凝胶混合物在室温下稳定至少两至三个月
  5. 5%变性凝胶(30ml) 5ml 30%丙烯酰胺/双丙烯酰胺 25 ml凝胶混合物
    300微升10%APS
    30μlTEMED
  6. 5%无变性凝胶
    6.75ml 40%丙烯酰胺/双丙烯酰胺(38:2) 4.5 ml的10x TBE
    300μl10%APS
    30μl的TEMED
    33.6ml H 2 O 2 /
  7. TNS解决方案
    25mM Tris-HCl pH7.5 400 mM NaCl
    0.1%SDS
  8. 尿素凝胶的RNA加载染料
    20mM Tris-HCl pH7.6
    8 M尿素
    1mM EDTA
    0.05%二甲苯蓝 0.05%溴酚蓝
  9. 用于非尿素凝胶的6x装载缓冲液
    30%甘油
    0.3%溴酚蓝
    0.3%二甲苯基氰
  10. 装订/清洗缓冲区
    20mM Tris-HCl,pH7.5 150mM NaCl 0.1%Triton X-100 1x蛋白酶抑制剂
  11. 磷酸盐缓冲液
    10mM Na 2 HPO 4
    2mM KH 2 PO 4 sub/
    2.7 mM KCl
    137 NaCl pH7.4
  12. 柠檬酸盐缓冲液
    2 M柠檬酸
    用10M KOH调节pH至2,稀释至0.2M

致谢

该协议改编自Thomas 等人(2012)。 这项工作得到了美国国家科学基金会的资助。

参考文献

  1. Day,I.S.,Golovkin,M.,Palusa,S.G.,Link,A.,Ali,G.S.Thomas,J.,Richardson,D.N.and Reddy,A.S。(2012)。 SR-like蛋白与剪接体蛋白的相互作用SR45 内含子序列:了解调节剪接。

    71(6):936-947。
  2. Golovkin,M。和Reddy,A.S。(1999)。 SC35样蛋白和一种新的丝氨酸/富含精氨酸蛋白与拟南芥U1-70K蛋白相互作用.33 J.Biol.Chem.274(51):36428-36438。
  3. Palusa,S. G.和Wilusz,J。(2013)。 RNA-蛋白质相互作用的鉴定和表征的方法。 Biophysical approach to translation control of gene expression,Biophysics for the Life Sciences,J.D.Dinman(eds)。 Springer:199-212。
  4. Ryder,S.P.,Recht,M.I。和Williamson,J.R。(2008)。 通过凝胶迁移率变化对蛋白质-RNA相互作用的定量分析方法Mol Biol 488: 99-115。 
  5. Thomas,J.,Palusa,S.G.,Prasad,K.V.,Ali,G.S.,Surabhi,G.K.,Ben-Hur,A.,Abdel-Ghany,S.E.and Reddy,A.S。(2012)。 识别 内含子剪接调节元件参与SCL33前mRNA的可变剪接的自动调节。植物J72(6):935-946。
  6. Wilusz,J。和Shenk,T。(1988)。 一个64 kd核蛋白结合到包含AAUAAA多腺苷酸化基序的RNA片段。 em> Cell 52(2):221-228。
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引用:Palusa, S. G. and Reddy, A. S. (2013). Analysis of RNA-protein Interactions Using Electrophoretic Mobility Shift Assay (Gel Shift Assay). Bio-protocol 3(22): e967. DOI: 10.21769/BioProtoc.967.
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Sung Ki Cho
Iowa State university
Hi, I am experiencing signal on the well position. Notes 12 recommends to use BSA to minimize it, but how about the concentration of BSA to improve the issue? Thank you in advance.

SK
8/27/2014 12:59:12 PM Reply
saiprasad palusa
Department of Biology, Colorado State University, USA

Hi,
we normally use 100ug/ml final concentration of BSA for this, but based on your conditions you have to optimize this.
And also your protein should be purified as much as possible
Not exceed more than 15ul of loading volume, if you increase the loading volume, you are increasing the salt concentration and this will effect on PH and finally it will create problem on running condition. If possible add 1 ul tRNA in addition Heparin sulfate.
good luck

8/28/2014 8:52:09 AM


Juseok Lee
Seoul National University
Is there any big difference between DNA-EMSA and RNA-EMSA based on your experience?
I'm doing some RNA-EMSA exp. as screening and not that successful so far.

I need to confirm that this result come from no interaction between RNA sequence and protein or my procedure problem.

Thanks in advance.

LJS
2/26/2014 3:19:17 AM Reply
Bio-protocol Editorial Team
bio-protocol.org

Hi Juseok,

Sorry to take so long to get back to you. Your above question seems to be a kind of general question. We would suggest that more specific question/comment related to this protocol could be addressed more effectively by our authors or other experts.

Thanks,
Bio-protocol Editorial Board

3/20/2014 1:35:27 PM


saiprasad palusa
Department of Biology, Colorado State University, USA
Do you have any experience with poly[dI-dC], instead of heprain sulfate?
poly[dI-dC] commonly used in DNA-EMSA & I just wonder whether it could work in RNA-EMSA or not.
Or is there any specific reason to use heparin sulfate?"

Hi,
we never used poly[dI-dC] for RNA -EMSA, since five years we are using heparin sulfate and it is working very well. Heparin sulfate used for reduce the non specific binding like poly[dI-dC] in DNA-EMSA.

Thanks
2/18/2014 8:20:44 AM Reply
Juseok Lee
Seoul National University
Hi,

Do you have any experience with poly[dI-dC], instead of heprain sulfate?
poly[dI-dC] commonly used in DNA-EMSA & I just wonder whether it could work in RNA-EMSA or not.
Or is there any specific reason to use heparin sulfate?
2/18/2014 2:37:31 AM Reply
Bio-protocol team Bio-protocol team
bio-protocol

Hi,

Please see Prof. Palusa's response to your question above.

Thanks,

Bio-protocol Editorial Borad

2/21/2014 5:56:17 PM