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Protein-RNA ELISA Assay
蛋白质-RNA酶联免疫吸附试验

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Abstract

Protein-RNA ELISA assay is an effective and quantitative method to study protein-RNA interactions in vitro. In this protocol we used recombinant 6x HIS tagged protein, but it works as well for non tagged proteins.

Keywords: RNA(RNA), Protein(蛋白), Interaction(相互作用), RNA-ELISA(rna-elisa)

Materials and Reagents

  1. 96-well Microplate Nunc maxisorp White (Thermo Fisher Scientific, Nunc, catalog number: 436110 )
  2. Recombinant 6x HIS tagged protein
  3. Streptavidin (New England Biolabs, catalog number: N7021S )
  4. NaHCO3
  5. Phosphate buffered saline (PBS) (Life Technologies, Gibco®, catalog number:  10010031 )
  6. Tween 20
  7. BSA
  8. Biotinylated RNA
  9. 1 M Tris (pH 7.5)
  10. NaCl
  11. Yeast tRNA (Life Technologies, Applied Biosystems®, catalog number:  AM7119 )
  12. 6x His mAb-HRP Conjugate (Clontech, catalog number: 631210 )
  13. ECL Peroxidase Substrate Solution A and B (Pierce Antibodies, catalog number:  32106 )
  14. Water used for all solution is RNAase free
  15. q-PCR tape (R&D systems, catalog number: DY992 )
  16. RiboLoc RNAase Inhibitor (Thermo Fisher Scientific, catalog number:  EO0381 )
  17. RNA 3' End Biotinylation Kit (Pierce Antibodies, catalog number: 20160
  18. Binding buffer (see Recipes)
  19. PBS-T (see Recipes)

Equipment

  1. Luminescence Plate reader (BMG LABTECH, FLUOstar OPTIMA)
  2. Thermocycler
  3. Centrifuges

Procedure

  1. Coated 96-well plates with 50 μl/well of streptavidin (100 μg/ml in 0.1 M NaHCO3).
  2. Incubate overnight at 4 °C. Seal the wells with q-PCR tape.
  3. Wash plates six times with 200 μl PBS-T (there is no need to incubate for washing).
  4. Block with 50 μl/well of PBS containing 3% BSA and 0.1 μg/ml streptavidin.
  5. Incubate overnight at 4 °C or 7 h at RT. Seal the wells with q-PCR tape (for my experience, in this step overnight or at least 7 h RT incubation is needed as less time causes high noise).
  6. Mix in pcr tubes biotinylated RNA (5 pmol) and different amounts of protein (0-500 ng) in binding buffer. The final volume in each tube is 50 μl. The RNA was biotinylated using the RNA 3' End Biotinylation Kit.
  7. Incubate the pcr tubes with the biotinylated RNA plus the protein, in a thermocycler for 30 min at 37 °C.
  8. Wash plates four times with 200 μl PBS-T.
  9. Transfer the mix (biotinylated RNA-protein) in to the plates (50 μl/well) and incubate 1 h at RT. Seal the wells with q-PCR tape (No need to rock to rock/shake the plate).
  10. Wash plates six times with 200 μl/well PBS-T.
  11. Add the 6x His mAB/HRP conjugate in PBS 1: 1,000 (50 μl/well) incubated for 1 h at RT. Seal the wells with q-PCR tape
  12. Wash plates six times with 200 μl/well PBS-T.
  13. Add 50 μl/well of a mix 1:1 ECL (25 μl of ECL1 and 25 μl of ECL2).
  14. Spin the plate to avoid bubbles using an adaptor for a swing bucket centrifuge.
  15. Read luminescence in the Labtech FLUOstar OPTIMA (MARS Software from labtech on Nunc maxisorp 96. 0.5 second interval).

Recipes

  1. Binding buffer
    50 mM Tris (pH 7.5)
    150 mM NaCl
    0.02 mg/ml yeast tRNA
    0.2 mg/ml BSA
    1.5 μl RiboLoc RNAase Inhibitor
    RNAase free water
    In a total volume of 50 μl per well
  2. PBS-T
    1x PBS
    0.1% Tween 20

Acknowledgments

This protocol was adapted from Gajjar et al. (2012). This work was supported by INSERM, La Ligue Contre le Cancer, and RECAMO CZ.1.05/2.1.00/03.0101. M.M.C. was supported by JSPS, AXA Research Fund, and Fundacao para a Ciencia e Tecnologia of Portugal. M.G. was supported by a research fellowship from Indo-French Centre for the Promotion of Advanced Research (IFCPAR) and from the ARC.

References

  1. Gajjar, M., Candeias, M. M., Malbert-Colas, L., Mazars, A., Fujita, J., Olivares-Illana, V. and Fahraeus, R. (2012). The p53 mRNA-Mdm2 interaction controls Mdm2 nuclear trafficking and is required for p53 activation following DNA damage. Cancer Cell 21(1): 25-35.
  2. MacCallum, D. E. and Hupp, T. R. (1999). Induction of p53 protein as a marker for ionizing radiation exposure in vivo. Methods Mol Biol 113: 583-589.

简介

蛋白质-RNA ELISA测定法是一种用于体外研究蛋白质-RNA相互作用的有效和定量方法。 在这个协议中,我们使用重组6x HIS标记蛋白,但它也适用于非标记蛋白。

关键字:RNA, 蛋白, 相互作用, rna-elisa

材料和试剂

  1. 96孔微孔板Nunc maxisorp White(Thermo Fisher Scientific,Nunc,目录号:436110)
  2. 重组6x HIS标签蛋白
  3. 链霉亲和素(New England Biolabs,目录号:N7021S)
  4. NaHCO 3
  5. 磷酸盐缓冲盐水(PBS)(Life Technologies,Gibco ,目录号:10010031)
  6. 吐温20
  7. BSA
  8. 生物素化RNA
  9. 1M Tris(pH7.5)
  10. NaCl
  11. 酵母tRNA(Life Technologies,Applied Biosystems ,目录号:AM7119)
  12. 6x His mAb-HRP缀合物(Clontech,目录号:631210)
  13. ECL过氧化物酶底物溶液A和B(Pierce抗体,目录号:32106)
  14. 用于所有溶液的水不含RNA酶
  15. q-PCR带(R& D systems,目录号:DY992)
  16. RiboLoc RNA酶抑制剂(Thermo Fisher Scientific,目录号:EO0381)
  17. RNA 3'端生物素化试剂盒(Pierce Antibodies,目录号:20160)
  18. 结合缓冲液(参见配方)
  19. PBS-T(参见配方)

设备

  1. 发光酶标仪(BMG LABTECH,FLUOstar OPTIMA)
  2. 热循环仪
  3. 离心机

程序

  1. 用50μl/孔的链霉亲和素(100μg/ml,在0.1M NaHCO 3中)包被96孔板。
  2. 在4℃孵育过夜。 用q-PCR胶带密封孔。
  3. 用200μlPBS-T洗涤板6次(无需孵育用于洗涤)。
  4. 用50μl/孔的含有3%BSA和0.1μg/ml链霉亲和素的PBS封闭。
  5. 在4℃孵育过夜或在室温下孵育7小时。 用q-PCR胶带密封孔(对于我的经验,在这一步骤过夜或至少7小时RT孵化需要较少的时间导致高噪声)。
  6. 在结合缓冲液中混合生物素化的RNA(5pmol)和不同量的蛋白质(0-500ng)。 每个管中的最终体积为50μl。 使用RNA 3'端生物素化试剂盒将RNA生物素化
  7. 孵育pcr管与生物素化的RNA加上蛋白质,在热循环仪在37℃下30分钟。
  8. 用200μlPBS-T洗涤板四次。
  9. 将混合物(生物素化的RNA-蛋白)转移到板(50μl/孔)中,并在室温下孵育1小时。 用q-PCR胶带密封孔(不需要摇动摇动/摇动平板)。
  10. 用200μl/孔PBS-T洗涤板六次。
  11. 加入PBS中的6x His mAB/HRP缀合物1:1,000(50μl/孔),在室温下温育1小时。 用q-PCR胶带密封孔
  12. 用200μl/孔PBS-T洗涤板六次。
  13. 加入50μl/孔的混合1:1 ECL(25μl的ECL1和25μl的ECL2)。
  14. 旋转板,以避免气泡使用适配器的秋千桶离心机。
  15. 在Labtech FLUOstar OPTIMA(MARS软件,Labtech在Nunc maxisorp 96. 0.5秒间隔)上读取发光。

食谱

  1. 绑定缓冲区
    50mM Tris(pH7.5) 150mM NaCl 0.02毫克/毫升酵母tRNA
    0.2 mg/ml BSA
    1.5μlRiboLoc RNA酶抑制剂
    无RNA酶水
    总体积为50μl/孔
  2. PBS-T
    1x PBS
    0.1%Tween 20

致谢

该方案改编自Gajjar等人(2012)。 这项工作得到INSERM,La Ligue Contrele Cancer和RECAMO CZ.1.05/2.1.00/03.0101的支持。 M.M.C. 得到JSPS,AXA研究基金和Fundacao para a 葡萄牙的Ciencia e技术。 M.G. 得到了印度 - 法国促进高级研究中心(IFCPAR)和ARC的研究奖学金的支持。

参考文献

  1. Gajjar,M.,Candeias,M.M.,Malbert-Colas,L.,Mazars,A.,Fujita,J.,Olivares-Illana,V.and Fahraeus,R。(2012)。 p53 mRNA-Mdm2相互作用控制Mdm2核运输,是DNA损伤后p53活化所必需的。/a> Cancer Cell 21(1):25-35。
  2. MacCallum,D.E。和Hupp,T.R。(1999)。 在体内诱导p53蛋白作为电离辐射暴露的标记/a>。 Methods Mol Biol 113:583-589
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引用:Illana, V. O. and Farhaeus, R. (2012). Protein-RNA ELISA Assay. Bio-protocol 2(17): e257. DOI: 10.21769/BioProtoc.257.
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