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RNA-Affinity Chromatography
RNA亲和色谱法   

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Abstract

RNA-affinity chromatography assays are used to identify proteins binding specific RNA sequences. These proteins represent potential factors contributing to the function of RNA molecules. In our lab, we have used this protocol to identify proteins binding sequence motifs involved in replication and transcription of positive strand RNA viruses. The assay described in this protocol consists on the immobilization of 5’-biotinylated RNA oligonucleotides (30-40 nt) on a streptavidin-conjugated, paramagnetic solid matrix. Then, cytoplasmic protein extracts pre-cleared on the solid matrix to decrease nonspecific binding, were incubated with the immobilized RNA molecules in the presence of a nonspecific competitor. RNA-protein complexes immobilized on the paramagnetic solid matrix were isolated using a magnet and the bound proteins were separated by polyacrylamide gel electrophoresis for proteomic analysis.

Keywords: RNA-protein interactions(RNA和蛋白质的相互作用), Biotinylated-RNA(生物素标记的RNA), Biotin-streptavidin interaction(生物素-相互作用), Paramagnetic solid matrix(顺磁性固体基质)

Materials and Reagents

  1. 5’-biotinylated RNAs (Sigma-Aldrich)
  2. Streptavidin conjugated solid matrix (Dynabeads M-280 Streptavidin) (Life Technologies, InvitrogenTM, catalog number: 11205D )
  3. Protease inhibitor (Complete Protease Inhibitor Cocktail Tablets) (Roche, catalog number: 1697498 )
  4. 10% glycerol
  5. Non-specific competitor tRNA (Baker yeast) (Sigma-Aldrich, catalog number: R8759 )
  6. NuPAGE LDS sample buffer (Life Technologies, InvitrogenTM)
  7. Dithiothreitol (DTT) (Sigma-Aldrich, catalog number: D9779 )
  8. Bis-Tris-Gel (Life Technologies, InvitrogenTM)
  9. NuPAGE MOPS SDS Running Buffer (Life Technologies, InvitrogenTM)
  10. Coomassie Simply Blue Safe Stain (Life Technologies, InvitrogenTM, catalog number: LC6060 )
  11. Diethylpyrocarbonate (DEPC) treated water
  12. RNase inhibitor (RNasin) (Promega, catalog number: N2611 )
  13. IGEPAL CA-630 (NP-40 substitute) (Sigma-Aldrich, catalog number: I3021 )
  14. KCl
  15. Glycerol
  16. NP-40
  17. EDTA
  18. Extraction buffer (see Recipes)
  19. H-BW solution (see Recipes)
  20. BW solution (see Recipes)

Equipment

  1. 150 mm plates
  2. Protein gel cassettes
  3. Orbital Shaker (J.P. SELECTA, catalog number: Orbit 3000445 )
  4. Magnetic particle concentrator for microcentrifuge tubes (DYNAL BIOTECH, Dynal MPC-S 120.20)

Software

  1. Image Lab V3.0 (Bio-Rad)

Procedure

  1. The capture of proteins binding specific RNA was performed using 5’-biotinylated RNAs linked to a streptavidin conjugated solid matrix.
  2. Proteins were extracted from transmissible gastroenteritis virus (TGEV) infected human Huh7 cells using an extraction buffer containing 10% Igepal (NP-40) detergent. The cell lysate was then centrifuged to pellet the nuclei and save the cytoplamic extract. Briefly, Huh7 cells grown on 150 mm plates were infected with TGEV virus. At 48 h post infection, cells from two plates were harvested and resuspended with 500 μl extraction buffer without Igepal (NP-40) on ice. Cellular extracts were incubated for 15 min on ice and then, Igepal (NP-40) detergent was added to the cell suspension to a final concentration of 10%. Extracts were mixed by vortexing, incubated for 10 additional minutes on ice and centrifuged for 2 min at maximum speed to recover the supernatant. Protein extracts may be storaged at -80 °C with 10% glycerol.
  3. 60 μl of streptavidin-conjugated solid matrix (10 μg μl-1) were used per RNA binding assay. Before binding to RNA, the solid matrix was washed twice with 360 μl of solution H-BW. All washes were performed by inverting the tube. No incubation time was required. After washing, the solid matrix was separated from the supernatant using a magnet. Leave the tubes in the magnet for 1-2 min.
  4. Pre-clearing protein extracts on solid matrix not bound to RNA. Protein extracts diluted in H-BW solution (500 μg of total protein per RNA binding assay), were precleared three times by incubating with 60 μl of solid matrix in an orbital shaker at 12 rpm, for at least 5 h each time at 4 °C. Separate the solid matrix using a magnet. Leave the tubes in the magnet for 1-2 min. The solid matrix was discarded after each pre-clearing incubation and the supernantant was transferred to a new tube containing 60 μl of new solid matrix. After the third preclearing, the supernatant was preserved for RNA-binding (step 7).
  5. RNA-immobilization on the streptavidin solid matrix. For each RNA binding assay, 60 μl of solid matrix were used. Previously, the solid matrix was washed twice with 60 μl of BW solution as a minimum volume. Then, the streptavidin matrix was incubated with the biotinylated RNA (8 μg) in 60 μl of BW solution for 30 min at RT.
  6. Immobilized RNAs on the solid matrix were washed twice with 60 μl of H-BW solution as a minimum volume. Remove the wash solution after placing the tubes in a magnet.
  7. RNA-protein binding. Add to the immobilized RNAs, 500 μg of precleared protein extract (from step 4) resuspended in H-BW solution and different amounts of non-specific competitor tRNA (0.5 or 1.25 μg tRNAs μg-1 protein). Incubate the mixture overnight in an orbital shaker at 4 °C and a speed of 12 rpm. The total volume of the sample was 180 μl (three times the solid matrix volume) consisting of 120 μl of precleared protein extract, 25 μl of tRNAs and 35 μl of H-BW. All the solutions were prepared in DEPC-water in the presence of 0.4 U/μl of RNAse inhibitor to minimize RNA degradation.
  8. Place the tubes in a magnet and remove the supernatant containing non-bound proteins. Wash three times with 120 μl H-BW solution.
  9. Elute the proteins bound to immobilized RNAs with NuPAGE LDS sample buffer supplemented with DTT 100 mM for 10 min at RT.
  10. Proteins bound to RNAs were resolved in NuPAGE 4-12% Bis-Tris-Gel by electrophoresis with NuPAGE MOPS SDS running buffer. Finally gels were stained with Coomassie Simply Blue Safe Stain. Images were taken with Image Lab V3.0.

Recipes

  1. Extraction buffer
    2.5 mM Hepes pH 7.9
    2.5 mM KCl
    25 μM EDTA
    0.25 mM DTT
    Protease inhibitor: One tablet diluted in 25 ml extraction buffer.
    Igepal is added (if needed) to a final concentration of 10%.
  2. H-BW solution
    50 mM HEPES pH 7.9
    150 mM KCl
    5% glycerol
    0.01% NP-40
  3. BW solution
    5 mM Tris HCl (pH 7.5)
    1 mM EDTA
    1 M NaCl

Acknowledgments

This work was supported by grants from the Ministry of Science and Innovation of Spain (BIO2010-167075) and the European Community’s Seventh Framework Programme (FP7/2007-2013), under the project PoRRSCon (EC grant agreement 245141). L.M. and P.A.M.-G. received a predoctoral fellowship from the Ministry of Science and Innovation of Spain (BES-2011-043489 and BES-2008-001932, respectively). We gratefully acknowledge C. M. Sanchez, and M. Gonzalez for technical assistance.

References

  1. Mateos-Gomez, P. A., Morales, L., Zuniga, S., Enjuanes, L. and Sola, I. (2013). Long-distance RNA-RNA interactions in the coronavirus genome form high-order structures promoting discontinuous RNA synthesis during transcription. J Virol 87(1): 177-186.

简介

RNA亲和层析分析用于鉴定结合特异性RNA序列的蛋白质。 这些蛋白质代表有助于RNA分子功能的潜在因素。 在我们的实验室,我们使用这个方案来鉴定参与正链RNA病毒复制和转录的蛋白质结合序列基序。 该方案中描述的测定法包括将5'-生物素化的RNA寡核苷酸(30-40nt)固定在链霉抗生物素蛋白缀合的顺磁性固体基质上。 然后,在固体基质上预先清除的细胞质蛋白质提取物以降低非特异性结合,在非特异性竞争剂存在下与固定的RNA分子一起温育。 使用磁铁分离固定在顺磁性固体基质上的RNA-蛋白复合物,并通过聚丙烯酰胺凝胶电泳分离结合蛋白进行蛋白质组学分析。

关键字:RNA和蛋白质的相互作用, 生物素标记的RNA, 生物素-相互作用, 顺磁性固体基质

材料和试剂

  1. 5'-生物素化RNA(Sigma-Aldrich)
  2. 链霉抗生物素复合固体基质(Dynabeads M-280链霉亲和素)(Life Technologies,Invitrogen TM,目录号:11205D)
  3. 蛋白酶抑制剂(Complete Protease Inhibitor Cocktail Tablets)(Roche,目录号:1697498)
  4. 10%甘油
  5. 非特异性竞争者tRNA(Baker酵母)(Sigma-Aldrich,目录号:R8759)
  6. NuPAGE LDS样品缓冲液(Life Technologies,Invitrogen TM )
  7. 二硫苏糖醇(DTT)(Sigma-Aldrich,目录号:D9779)
  8. Bis-Tris-Gel(Life Technologies,Invitrogen TM )
  9. NuPAGE MOPS SDS运行缓冲液(Life Technologies,Invitrogen TM )
  10. 考马斯简单蓝色安全污渍(Life Technologies,Invitrogen TM ,目录号:LC6060)
  11. 碳酸二乙酯(DEPC)处理水
  12. RNase抑制剂(RNasin)(Promega,目录号:N2611)
  13. IGEPAL CA-630(NP-40替代品)(Sigma-Aldrich,目录号:I3021)
  14. KCl
  15. 甘油
  16. NP-40
  17. EDTA
  18. 提取缓冲液(见配方)
  19. H-BW解决方案(见配方)
  20. BW解决方案(见配方)

设备

  1. 150毫米板
  2. 蛋白胶盒
  3. 轨道震荡器(J.P.SELECTA,目录号:Orbit 3000445)
  4. 微量离心管用磁粉浓缩机(DYNAL BIOTECH,Dynal MPC-S 120.20)

软件

  1. Image Lab V3.0(Bio-Rad)

程序

  1. 使用与链霉亲和素缀合的固体基质连接的5'-生物素化的RNA来进行蛋白质结合特异性RNA的捕获。
  2. 使用含有10%Igepal(NP-40)洗涤剂的提取缓冲液从传染性胃肠炎病毒(TGEV)感染的人Huh7细胞中提取蛋白质。然后将细胞裂解物离心以沉淀核并保存细胞质提取物。简言之,在150mm平板上生长的Huh7细胞用TGEV病毒感染。在感染后48小时,收获来自两个平板的细胞,并用500μl不含Igepal(NP-40)的提取缓冲液重悬于冰上。将细胞提取物在冰上孵育15分钟,然后将Igepal(NP-40)洗涤剂加入到细胞悬液中至终浓度为10%。通过涡旋将提取物混合,在冰上再孵育10分钟,并以最大速度离心2分钟以回收上清液。蛋白质提取物可以在-80℃下用10%甘油储存。
  3. 每个RNA结合测定使用60μl链霉抗生物素蛋白缀合的固体基质(10μgμl -1 )。在结合RNA之前,用360μl溶液H-BW洗涤固体基质两次。所有洗涤都是通过翻转管进行的。不需要孵化时间。洗涤后,使用磁铁将固体基质与上清液分离。将管放在磁铁中1-2分钟。
  4. 将未结合RNA的固体基质上的蛋白质提取物预清除。通过在轨道振荡器中以12rpm的速度与60μl固体基质一起温育至少5h,每次在4°时预先清除在H-BW溶液中稀释的蛋白质提取物(每个RNA结合测定法500μg的总蛋白)三次C。用磁铁分离固体基质。将管放在磁铁中1-2分钟。每个预清洗孵育后弃去固体基质,并将上清液转移到含有60μl新固体基质的新管中。第三次预清除后,保留上清液进行RNA结合(步骤7)
  5. RNA固定在链亲和素固体基质上。对于每个RNA结合测定,使用60μl固体基质。以前,固体基质用最小体积的60μlBW溶液洗涤两次。然后,将链霉抗生物素蛋白基质与60μlBW溶液中的生物素化RNA(8μg)在室温下孵育30分钟。
  6. 将固体基质上固定的RNA作为最小体积用60μlH-BW溶液洗涤两次。将管置于磁铁中后,取出洗涤溶液。
  7. RNA蛋白结合。加入固定化的RNA,将500μg预先清除的蛋白质提取物(来自步骤4)重新悬浮于H-BW溶液和不同量的非特异性竞争剂tRNA(0.5或1.25μgtRNAsμg -1蛋白) 。在4℃和12rpm的速度下,在轨道振荡器中将混合物孵育过夜。样品的总体积为180μl(固体基质体积的三倍),由120μl预清蛋白提取物,25μltRNA和35μlH-BW组成。所有溶液在DEPC-水中,在0.4U /μlRNA酶抑制剂存在下制备,以使RNA降解最小化。
  8. 将管置于磁铁中,除去含有非结合蛋白的上清液。用120μlH-BW溶液洗三次
  9. 用补充有DTT 100mM的NuPAGE LDS样品缓冲液在室温下洗脱与固定的RNA结合的蛋白质10分钟。
  10. 通过与NuPAGE MOPS SDS运行缓冲液电泳,结合RNAs的蛋白质在NuPAGE 4-12%Bis-Tris-Gel中解析。最后凝胶用考马斯Simply Blue Safe Stain染色。图像采用Image Lab V3.0。

食谱

  1. 提取缓冲区
    2.5 mM Hepes pH 7.9
    2.5 mM KCl
    25μMEDTA
    0.25 mM DTT
    蛋白酶抑制剂:将一片稀释于25ml提取缓冲液中 添加Igepal(如果需要)至最终浓度为10%。
  2. H-BW解决方案
    50 mM HEPES pH 7.9
    150 mM KCl
    5%甘油
    0.01%NP-40
  3. BW解决方案
    5 mM Tris HCl(pH 7.5)
    1 mM EDTA
    1 M NaCl

致谢

这项工作得到了西班牙科学与创新部(BIO2010-167075)和欧洲共同体第七框架计划(FP7 / 2007-2013)在项目PoRRSCon(EC授权协议245141)下的资助。 L.M.和P.A.M.-G. 获得了西班牙科学与创新部(BES-2011-043489和BES-2008-001932)的前瞻性奖学金。 我们衷心感谢C. M. Sanchez和M. Gonzalez的技术援助。

参考

  1. Mateos-Gomez,PA,Morales,L.,Zuniga,S.,Enjuanes,L.and Sola,I.(2013)。87(1):177-186。
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Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
引用:Morales, L., Mateos-Gomez, P. A., Enjuanes, L. and Sola, I. (2013). RNA-Affinity Chromatography. Bio-protocol 3(13): e808. DOI: 10.21769/BioProtoc.808.
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