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RNA-binding Protein Immunoprecipitation (RIP) to Examine AUF1 Binding to Senescence-Associated Secretory Phenotype (SASP) Factor mRNA
RNA结合蛋白免疫共沉淀法(RIP)检验结合到衰老相关分泌表型(SASP)因子 的mRNAAUF1   

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Abstract

Immunoprecipitation and subsequent isolation of nucleic acids allows for the investigation of protein:nucleic acid interactions. RNA-binding protein immunoprecipitation (RIP) is used for the analysis of protein interactions with mRNA. Combining RIP with quantitative real-time PCR (qRT-PCR) further enhances the RIP technique by allowing for the quantitative assessment of RNA-binding protein interactions with their target mRNAs, and how these interactions change in different cellular settings. Here, we describe the immunoprecipitation of the RNA-binding protein AUF1 with several different factors associated with the senescence-associated secretory phenotype (SASP) (Alspach and Stewart, 2013), specifically IL6 and IL8. This protocol was originally published in Alspach et al. (2014).

Materials and Reagents

  1. Tissue culture
    1. DMEM (Sigma-Aldrich, catalog number: D6429 )

    2. Fetal bovine serum (FBS) (Sigma-Aldrich, catalog number: F2442 )
    3. Medium M199 (Sigma-Aldrich, catalog number: M7528 )
    4. Penicillin/streptomycin (Sigma-Aldrich, catalog number: P4333 )

    5. BJ human foreskin fibroblasts

    6. Bleomycin sulfate (Sigma-Aldrich, catalog number: B8416 )
    7. BJ culture media (see Recipes)

  2. General materials
    1. Bradford Protein Assay reagent (Bio-Rad Laboratories, catalog number: 500-0006 )
    2. Polyclonal anti-AUF1 antibody (EMD Millipore, catalog number: 07260MI )

    3. Normal Rabbit IgG antibody (Cell Signaling Technology, catalog number: 2729 )
    4. Protein A Dynabeads (Life Technologies, catalog number: 10002D )

    5. MCLB (mammalian cell lysis buffer) (see Recipes)
    6. NT2 buffer
(see Recipes)
    7. Buffer A (see Recipes)
    8. Buffer B (see Recipes)
    9. Buffer C (see Recipes)

  3. Immunoprecipitation reagents
    1. Tris

    2. EDTA

    3. NP40

    4. NaCl

    5. Phosphate-buffered saline (PBS)

    6. SDS

    7. Deoxycholate

    8. MgCl2
    9. NaH2PO4

    10. RNAse OUT (Life Technologies, catalog number: 10777-019 )

    11. Proteinase K (Sigma-Aldrich, catalog number: P6556 )
    12. RiboPure RNA isolation kit (Life Technologies, catalog number: AM1924 )

  4. qRT-PCR reagents
    1. dNTPs

    2. Hexanucleotide mix (Roche Diagnostics, catalog number: 11277081001 )
    3. SuperScript II Reverse Transcriptase (Life Technologies, catalog number: 18064-014 )
    4. Power SYBR GREEN PCR master mix (Life Technologies, catalog number: 4367659 )

  5. Primers
    1. IL6 forward: 5′-ACA TCCTCGACGGCA TCTCA-3′
    2. IL6 reverse: 5′-TCACCAGGCAAGTCTCCTCA-3′
    3. IL8 forward: 5′-GCTCTGTGTGAAGGTGCAGT-3′
    4. IL8 reverse: 5′-TGCACCCAGTTTTCCTTGGG-3′

Equipment

  1. Tissue Culture
    1. 37 °C forced-air incubator maintained at 5% CO2 and 5% O2 (Thermo Fisher Scientific, Forma Series II water jacketed CO2 incubator)
    2. Laminar-flow biosafety cabinet (Labconco Purifier Class II Biosafety Cabinet)

  2. Immunoprecipitation
    1. Spectrophotometer capable of reading at 450 and 595 nm
    2. Dyna-mag magnetic bead separator (Life Technologies, catalog number: 12321D )
    3. Micro centrifuge
    4. End-over-end tube rotator (Thermo Fisher Scientific, catalog number: 400110Q )

  3. qRT-PCR
    1. PCR machine (Bio-Rad Laboratories, model: C1000 thermo cycler )

    2. Real-time PCR detection system (Bio-Rad Laboratories, model: CFX96 Real Time System )

  4. General equipment
    1. Tissue culture cell scrapers

    2. Refrigerated micro centrifuge
    3. Heat block capable of reaching 70 °C

Procedure

  1. BJ human foreskin fibroblasts were cultured at 37 °C, 5% CO2, 5% O2 in BJ culture media. Stress-induced premature senescence (SIPS) was induced by treating the cells with 100 μg/ml bleomycin sulfate for 24 h. Bleomycin sulfate-containing media was replaced with fresh media following the 24 h treatment. Cells are senescent 3 days following treatment with bleomycin.
  2. Collect cells by scraping in PBS on ice (for a 15 cm culture dish, 4 ml of PBS was used). 3.5 million cells are required for each immunoprecipitation (IP) (IgG control and specific antibody). Cell number was determined using a hemocytometer.
  3. Pellet cells by centrifugation at 1,500 rpm for 5 min at 4 °C.
  4. To lyse cells, resuspend the pellet in 1 ml MCLB and 
incubate for 20 min at 4 °C with end-over-end rotation.
  5. Centrifuge cell lysates at maximum speed for 20 min at 4 °C to remove 
cellular debris. Remove 1 ml of cell lysate to a micro centrifuge tube.
  6. Remove 100 μl of protein lysate from each and place in a new Eppendorf tube. This will be used as the input for each sample.
  7. Quantification of protein concentration was performed using the Bradford assay following manufacturer’s protocols.
  8. Prepare the magnetic beads for immunoprecipitation.
    1. Wash 100 μl beads per IP three times in 0.1 M NaH2PO4 (for 
example: If 2 AUF1 immunoprecipitations and 2 IgG immunoprecipitations are to be performed, you will require 200 μl of beads for the AUF1 IPs and 200 μl of beads for the IgG IPs). Washes are performed by resuspending the magnetic beads, and then separating them from solution using a magnetic micro centrifuge tube rack.
    2. Resuspend washed beads in 500 μl 0.1 M NaH2PO4 and add 30 μg anti-AUF1 or IgG control antibody per IP (for example: If you are performing 2 AUF1 IPs, resuspend your beads in 500 μl of 0.1 M NaH2PO4 and add 60 μg of anti-AUF1 antibody).
    3. Incubate beads with antibodies at room temperature for at least 1 h with end-over-end rotation.
    4. Wash antibody-conjugated beads 3 times in buffer A by resuspending beads in buffer A and pulling them down using a magnetic micro centrifuge tube rack. Leave beads in buffer A until ready for IP.
  9. Pre-clear 3 mg of protein per IP by incubating with 20 μl non-antibody conjugated beads. Bring up to 1 ml volume with MCLB. Incubate for 30 min at 4 °C with end-over-end rotation.
  10. At the same time, resuspend antibody-conjugated beads in NT2 buffer. At this time you need to aliquot the beads for the individual IPs. For example, if you are preparing to do 2 AUF1 IPs, resuspend the beads in 200 μl NT2 buffer and separate them in to 2 tubes of 100 μl each. Incubate for 30 min at room temperature.
  11. Immunoprecipitate overnight:
    1. Remove the beads from the pre-cleared protein lysate using a 
magnetic micro centrifuge tube rack, keeping the protein lysate and discarding the beads.
    2. Remove the antibody-conjugated beads from NT2 buffer using a
magnetic micro centrifuge tube rack, keeping the beads and discarding the NT2 buffer.
    3. Resuspend antibody-conjugated beads in pre-cleared protein 
lysate.
    4. Place tubes on end-over-end rotator. Incubate at 4 °C overnight.
  12. Wash the beads twice in each of the following buffers by resuspending the beads and then separating them from solution using a magnetic micro centrifuge tube rack
    1. Buffer A
    2. Buffer B
    3. Buffer C

  13. Incubate beads for 30 min at 55 °C in 100 μl NT2 buffer supplemented with:
    1. 0.1% SDS
    2. 80 U RNAse OUT
    3. 30 μg proteinase K
  14. Extract the RNA from the IP’s and input samples by adding 1 ml of Trizol (included in the RiboPure RNA isolation kit) to the mixture described in step 12. Incubate the solution for 5 min at room temperature. Proceed with the RNA isolation protocol provided in the RiboPure RNA isolation kit. Elute the RNA in 24 μl H2O.
  15. The eluted RNA can now be used to generate cDNA for qRT-PCR.
    1. cDNA was synthesized using hexanucleotide primers and 
following the manufacturer’s protocol provided with Super Script II 
Reverse Transcriptase.
    2. PCR was performed with Power SYBR Green master mix following the manufacturer’s protocol. The annealing temperature used for the primers that are outlined in the Materials and Reagents section is 60 °C.

Representative data



Figure 1. Representative results. Panel (A) represents expected results. The % input observed for AUF1 immunoprecipitation is above that observed for IgG immunoprecipitation, indicating that the AUF1 antibody pulled down more protein (and RNA) than the non-specific IgG antibody. Panel (B) represents a negative result, where immunoprecipitation with the AUF1 antibody did not pull down more protein (and RNA) than the non-specific IgG antibody.

Notes

  1. Please see Figure 2 for a simplified experimental workflow.


    Figure 2. RNA Immunoprecipitation (RIP) workflow. Lysates from each treatment are divided into three groups: 1) Inputs, which are used to normalize to overall expression level of the RNAs of interest, 2) IgG immunoprecipita8ons, which are used to set the background level of immunoprecipitation, and 3) AUF1 immunoprecipita8ons, used to investigate the relative level of AUF1 binding to your RNA of interest.

Recipes

  1. BJ culture media
    DMEM

    15% heat-inactivated fetal bovine serum
    15% medium 199

    1% penicillin/streptomycin

  2. MCLB (mammalian cell lysis buffer)
    50 mM Tris (pH 8.0)
    5 mM EDTA

    0.5% NP-40

    100 mM NaCl

  3. NT2 buffer

    50 mM Tris (pH 7.4)

    150 mM NaCl

    1 mM MgCl2
  4. Buffer A

    1x PBS

    0.1% SDS

    0.3% deoxycholate

    0.3% NP-40
  5. Buffer B
    5x PBS

    0.1% SDS

    0.5% deoxycholate
    0.5% NP-40

  6. Buffer C

    50 mM Tris-HCl (pH 7.4)
    10 mM MgCl2

    0.5% NP-40

Acknowledgments

This work was supported by NIH 5 R01 CA130919 (SAS), NIH Cellular Biochemical and Molecular Sciences Predoctoral Training Grant T32 GM007067 (E A), and an American Cancer Society Research Scholar Award (to S. A. Stewart). We thank Dr. Nicholas Davidson (Washington University School of Medicine) for his technical support in the generation of this protocol.

References

  1. Alspach, E., Fu, Y. and Stewart, S. A. (2013). Senescence and the pro-tumorigenic stroma. Crit Rev Oncog 18(6): 549-558.
  2. Alspach, E., Flanagan, K. C., Luo, X., Ruhland, M. K., Huang, H., Pazolli, E., Donlin, M. J., Marsh, T., Piwnica-Worms, D., Monahan, J., Novack, D. V., McAllister, S. S. and Stewart, S. A. (2014). p38MAPK plays a crucial role in stromal-mediated tumorigenesis. Cancer Discov 4(6): 716-729.

简介

免疫沉淀和随后的核酸分离允许研究蛋白质:核酸相互作用。 RNA结合蛋白免疫沉淀(RIP)用于分析蛋白质与mRNA的相互作用。 结合RIP与定量实时PCR(qRT-PCR)通过允许RNA结合蛋白与其靶mRNA的相互作用的定量评估以及这些相互作用在不同细胞设置中如何变化来进一步增强RIP技术。 在这里,我们描述RNA结合蛋白AUF1与几种不同的因素与衰老相关的分泌表型(SASP)(Alspach和斯图尔特,2013年),特别是IL6和IL8相关的免疫沉淀。 该协议最初发表在Alspach等人(2014)。

材料和试剂

  1. 组织培养
    1. DMEM(Sigma-Aldrich,目录号:D6429)
    2. 胎牛血清(FBS)(Sigma-Aldrich,目录号:F2442)
    3. 培养基M199(Sigma-Aldrich,目录号:M7528)
    4. 青霉素/链霉素(Sigma-Aldrich,目录号:P4333)
    5. BJ人包皮成纤维细胞
    6. 硫酸博莱霉素(Sigma-Aldrich,目录号:B8416)
    7. BJ培养基(见Recipes)

  2. 一般资料
    1. Bradford蛋白质测定试剂(Bio-Rad Laboratories,目录号:500-0006)
    2. 多克隆抗AUF1抗体(EMD Millipore,目录号:07260MI)
    3. 正常兔IgG抗体(Cell Signaling Technology,目录号:2729)
    4. 蛋白A Dynabeads(Life Technologies,目录号:10002D)
    5. MCLB(哺乳动物细胞裂解缓冲液)(参见配方)
    6. NT2缓冲区(参见配方)
    7. 缓冲区A(参见配方)
    8. 缓冲液B(参见配方)
    9. 缓冲液C(参见配方)

  3. 免疫沉淀试剂
    1. Tris
    2. EDTA
    3. NP40
    4. NaCl
    5. 磷酸盐缓冲盐水(PBS)
    6. SDS
    7. 脱氧胆酸盐
    8. MgCl 2
    9. NaH 2 PO 4 sub
    10. RNAse OUT(Life Technologies,目录号:10777-019)
    11. 蛋白酶K(Sigma-Aldrich,目录号:P6556)
    12. RiboPure RNA分离试剂盒(Life Technologies,目录号:AM1924)

  4. qRT-PCR试剂
    1. dNTPs
    2. 六核苷酸混合物(Roche Diagnostics,目录号:11277081001)
    3. SuperScript II Reverse Transcriptase(Life Technologies,目录号:18064-014)
    4. Power SYBR GREEN PCR主混合物(Life Technologies,目录号:4367659)

  5. 引物
    1. IL6正向:5'-ACA TCCTCGACGGCA TCTCA-3'
    2. IL6反向:5'-TCACCAGGCAAGTCTCCTCA-3'
    3. IL8正向:5'-GCTCTGTGTGAAGGTGCAGT-3'
    4. IL8反转:5'-TGCACCCAGTTTTCCTTGGG-3'

设备

  1. 组织文化
    1. 37℃强制空气培养箱中保持在5%CO 2和5%O 2(Thermo Fisher   Scientific,Forma Series II水夹套CO 2培养箱)
    2. 层流生物安全柜(Labconco净化器II类生物安全柜)

  2. 免疫沉淀
    1. 能在450和595nm读数的分光光度计
    2. Dyna-mag磁珠分离器(Life Technologies,目录号:12321D)
    3. 微量离心机
    4. 端到端管旋转器(Thermo Fisher Scientific,目录号:400110Q)

  3. qRT-PCR
    1. PCR仪(Bio-Rad Laboratories,型号:C1000热循环仪)
    2. 实时PCR检测系统(Bio-Rad Laboratories,型号:CFX96实时系统)

  4. 一般设备
    1. 组织培养细胞刮刀
    2. 冷藏微量离心机
    3. 热块能达到70°C

程序

  1. BJ人包皮成纤维细胞在BJ培养基中在37℃,5%CO 2,5%O 2下培养。通过用100μg/ml硫酸博莱霉素处理细胞24小时诱导应激诱导的早衰(SIPS)。在24小时处理后,用新鲜培养基替换含有硫酸博莱霉素的培养基。在用博来霉素处理后3天,细胞衰老。
  2. 通过在冰上在PBS中刮擦来收集细胞(对于15cm培养皿,使用4ml PBS)。每次免疫沉淀(IP)(IgG对照和特异性抗体)需要350万个细胞。使用血细胞计数器测定细胞数。
  3. 通过在4℃下以1,500rpm离心5分钟来沉淀细胞。
  4. 为了裂解细胞,将沉淀重悬于1ml MCLB中,并在4℃下以末端旋转孵育20分钟。
  5. 离心细胞裂解液以最大速度在4℃下20分钟,以去除细胞碎片。删除1毫升的细胞裂解液到微量离心管。
  6. 从每个删除100微升蛋白裂解液,并放置在一个新的Eppendorf管。这将用作每个样本的输入。
  7. 根据制造商的方案使用Bradford测定法进行蛋白质浓度的定量
  8. 准备磁珠免疫沉淀。
    1. 在0.1M NaH 2 PO 4中洗涤100μl珠子/IP三次(例如: 如果要进行2次AUF1免疫沉淀和2次IgG免疫沉淀 执行,您将需要200微升的珠子为AUF1 IP和200微升 的IgG IPs珠子)。 通过重悬浮进行洗涤 磁珠,然后使用磁性将它们从溶液中分离 微离心管架。
    2. 重悬洗涤珠在500μl0.1   M NaH 2 PO 4和每IP添加30μg抗AUF1或IgG对照抗体(对于 示例:如果您正在执行2个AUF1 IP,请将您的珠子重新悬挂在500 μl的0.1M NaH 2 PO 4 4并加入60μg的抗AUF1抗体)。
    3. 用抗体在室温下孵育珠至少1小时,其中末端旋转。
    4. 洗涤抗体偶联珠3次在缓冲液A中重悬 珠子在缓冲液A中,并使用磁性微粒将其向下拉 离心管架。 留在缓冲液A的珠子,直到准备IP。
  9. 通过与20μl非抗体缀合的珠孵育,预清洁3mg蛋白质/IP。 使用MCLB达到1 ml体积。 在4℃下,在末端旋转下孵育30分钟。
  10. 同时,在NT2缓冲液中重悬抗体偶联的珠子。 此时,您需要为各个IP分配珠子。 例如,如果你准备做2 AUF1 IP,重悬在200微升NT2缓冲区的珠子,并将它们分开到2管的100微升每个。 在室温下孵育30分钟。
  11. 免疫沉淀过夜:
    1. 使用磁性从预先清除的蛋白裂解液中删除珠 微量离心管架,保持蛋白裂解液并丢弃 珠子。
    2. 从NT2缓冲液中取出抗体偶联的珠子 使用磁性微离心管架,保持珠子和 丢弃NT2缓冲区。
    3. 在预纯化的蛋白裂解液中重悬抗体偶联的珠子
    4. 将管放在端到端旋转器上。 在4℃孵育过夜。
  12. 在以下每种缓冲液中洗涤珠两次,方法是重悬细胞珠,然后使用磁性微量离心管试管架将其从溶液中分离。
    1. 缓冲区A
    2. 缓冲区B
    3. 缓冲液C.
  13. 在补充有:
    的100μlNT2缓冲液中在55℃孵育珠子30分钟
    1. 0.1%SDS
    2. 80 U RNAse OUT
    3. 30μg蛋白酶K
  14. 通过加入1ml Trizol(包括在RiboPure RNA分离试剂盒中)到步骤12中所述的混合物中从IP和输入样品中提取RNA。在室温下孵育溶液5分钟。 继续RiboPure RNA分离试剂盒中提供的RNA分离方案。 洗脱在24μlH 2 O中的RNA
  15. 洗脱的RNA现在可用于产生qRT-PCR的cDNA
    1. 使用六核苷酸引物合成cDNA 制造商的协议提供与Super Script II反向 转录酶。
    2. 用Power SYBR Green master进行PCR 按照制造商的方案混合。 退火温度 用于在材料和试剂中概述的引物 截面为60℃。

代表数据



图1.代表性结果。 小图(A)代表预期的结果。观察到的AUF1免疫沉淀的%输入高于IgG免疫沉淀观察到的百分比,表明AUF1抗体下降 比非特异性IgG抗体更多的蛋白质(和RNA)。 图(B)代表阴性结果,其中用AUF1抗体的免疫沉淀不比非特异性IgG抗体下拉更多的蛋白质(和RNA)。

笔记

  1. 请参见图2,了解简化的实验工作流程。


    图2.RNA免疫沉淀(RIP)工作流程。来自每种处理的裂解物被分成三组:1)输入,其用于标准化感兴趣的RNA的整体表达水平,2)IgG免疫沉淀 ,其用于设定免疫沉淀的背景水平,和3)AUF1免疫沉淀,用于研究AUF1与感兴趣的RNA的结合的相对水平。

食谱

  1. BJ文化媒体
    DMEM
    15%热灭活的胎牛血清 15%培养基199
    1%青霉素/链霉素
  2. MCLB(哺乳动物细胞裂解缓冲液)
    50mM Tris(pH8.0) 5 mM EDTA
    0.5%NP-40
    100 mM NaCl
  3. NT2缓冲区
    50mM Tris(pH7.4)
    150mM NaCl 1mM MgCl 2
  4. 缓冲区A
    1x PBS
    0.1%SDS
    0.3%脱氧胆酸盐 0.3%NP-40
  5. 缓冲区B
    5x PBS
    0.1%SDS
    0.5%脱氧胆酸盐 0.5%NP-40
  6. 缓冲区C
    50mM Tris-HCl(pH7.4) 10mM MgCl 2/
    0.5%NP-40

致谢

这项工作由NIH 5 R01 CA130919(SAS),NIH细胞生物化学和分子科学Predoctoral训练补助T32 GM007067(E A)和美国癌症协会研究学者奖(S.A.Stewart)支持。我们感谢Nicholas Davidson博士(华盛顿大学医学院)在生成本协议方面的技术支持。

参考文献

  1. Alspach,E.,Fu,Y。和Stewart,S.A。(2013)。 衰老和致肿瘤发生基质。 Crit Rev Oncog 18(6):549-558。
  2. Alspach,E.,Flanagan,KC,Luo,X.,Ruhland,MK,Huang,H.,Pazolli,E.,Donlin,MJ,Marsh,T.,Piwnica-Worms,D.,Monahan, ,DV,McAllister,SS和Stewart,SA(2014)。 p38MAPK在基质介导的肿瘤发生中起着至关重要的作用。/em> 4(6):716-729。
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Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
引用:Alspach, E. and Stewart, S. A. (2015). RNA-binding Protein Immunoprecipitation (RIP) to Examine AUF1 Binding to Senescence-Associated Secretory Phenotype (SASP) Factor mRNA. Bio-protocol 5(10): e1481. DOI: 10.21769/BioProtoc.1481.
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