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[Bio101] RNP-IP (Original protocol)-getting majority of RNA from RNA binding protein in nucleus
[Bio101] 核糖核蛋白-免疫共沉淀(原始方法)—从细胞核所包含的RNA结合蛋白中获得大部分的RNA   

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Abstract

Post-transcriptional regulation of gene expression is a ribonucleoprotein (RNP)-driven process, which involves RNA binding proteins (RBPs) and noncoding RNAs that regulate splicing, nuclear export, subcellular localization, mRNA stability and translation. mRNAs encoding proteins that function in a particular cell process or pathway can be found within a unique mRNP complex, which consists of mRNA and RNP. This provides valuable information regarding not only known components of a particular process or pathway, but importantly, leads to the identification of novel components representing potential therapeutic targets and biomarkers. In addition to those targets identified by pathway expansion, the specific RBPs (RNA binding proteina) regulating RNA functions may be potential therapeutic targets in their own right. RNP-IP is a technology that allows the isolation and identification of mRNAs, microRNAs and protein components of RNP complexes from cell extracts using antibodies to RBPs. Once purified, the RNAs present in the complex are analyzed to identify the target mRNAs using various molecular biology tools such as RT-PCR, gene expression analysis based on microarray technology (Chip analysis), or sequencing. Using this method, more RNA that is present in the nucleus can be obtained.

Materials and Reagents

  1. Normal Rabbit IgG
  2. High-salt solution
  3. RIP-certified antibody (NBL, catalog number depends on what do you want to target)
  4. Protease inhibitor (Molecular Biology)
  5. Aprotinin
  6. Leupeptin
  7. Phenylmethylsulfonyl fluoride (PMSF)
  8. RNase inhibitor (Life Technologies, Invitrogen™, catalog number: 10777-019 )
  9. Dithiothreitol (DTT) (reducing agent)
  10. Protein A beads (GE Healthcare Dharmacon, catalog number: 17-0780-01 ) or Protein G beads (Thermo Fisher Scientific, catalog number: 22852 )
  11. Ethanol (Molecular Biology)
  12. 2-Propanol (Molecular Biology)
  13. Nuclease-free PBS
  14. Isotype control IgG (if necessary)
  15. Kit components (see Recipes)
  16. Commercial reagent (see Recipes)
  17. Buffer preparation (see Recipes)
  18. Wash buffer (see Recipes)
  19. Precaution: Additional buffer preparation (see Recipes)
  20. Preparation of antibody-immobilized protein A or protein G agarose beads-RNP complex

Equipment

  1. Microcentrifuge capable of 15,000 x g
  2. Microcentrifuge tube (1.5 ml or 2 ml) (Nuclease-free) (recommendation; use low-adhesion tube for RIP-Assay)
  3. Centrifuge capable of 2,000 x g
  4. Centrifuge tube (15 ml or 50 ml)
  5. Pipette (5 ml, 10 ml, 25 ml) (nuclease-free)
  6. Pipette tip (10 μl, 20-100 μl , 200 μl , and 1,000 μl) (nuclease-free) (recommendation; use low-adhesion pipette tip for RIP-Assay)
  7. Ultra low temperature freezer (-80 °C)
  8. Freezer (below -20 °C)
  9. End-over-end rotator
  10. Vortex mixer
  11. Gloves

Procedure

  1. Pre-step: Preparation of antibody-immobilized protein A or protein G agarose beads.
    1. Wash the protein A or protein G agarose beads 3 times with equal amount of nuclease-free PBS (centrifuge; 2,000 x g for 1 min at 4 °C).
    2. Aliquot 25 μl of the 50% beads slurry to each new microcentrifuge tube.
    3. Add 1 ml of wash buffer (+) to each tube.
    4. Add 15 μg of antibody (normal Rabbit IgG as a negative control or RIP-Certified antibody for target RBP, respectively) to each tube.
    5. Incubate the tube with rotation for at least 30 min at 4 °C. If necessary, this incubation can be extended to overnight.

  2. Pre-step: Preparation of protein A or protein G agarose beads for preclear
    1. Wash the protein A or protein G agarose beads 3 times with equal amount of nuclease-free PBS (centrifuge; 2,000 x g for 1 min at 4 °C).
    2. Aliquot 25 μl of the 50% beads slurry to each new microcentrifuge tube.
    3. Add 500 μl of wash buffer (+) to each tube, and mix briefly.
    4. Centrifuge the tube at 2,000 x g for 1 min at 4 °C.
    5. Discard the supernatant carefully.
    6. Leave the beads at 4 °C or on ice until starting Preclear step.
    7. Just before Preclear step, wash the beads once with 500 μl of lysis buffer (+).
    8. Centrifuge the tube at 2,000 x g for 1 min at 4 °C.
    9. Discard the supernatant carefully. Use these protein A or protein G agarose beads washed once with lysis buffer (+) for preclear step (step 28).

  3. Lysis of mammalian cells
    Note: In order to obtain “high-quality RNA”, freshly cultured cells should be used in RIP-Assay.
    1. Detach the cells from the culture dish by pipetting or using a cell scraper, if necessary. Collect the cell suspension into centrifuge tube.
    2. Centrifuge the cell suspension at 300 x g for 5 min at 4 °C to pellet the cells. Carefully remove and discard the supernatant.
    3. Wash the cells by resuspending the cell pellet with ice-cold PBS.
    4. Centrifuge the cell suspension at 300 x g for 5 min at 4 °C to pellet the cells. Carefully remove and discard the supernatant.
    5. Wash the cells once again using steps 17-18.
    6. Wash the cells by resuspending the cell pellet with ice-cold nuclease-free PBS.
    7. Centrifuge the cell suspension at 300 x g for 5 min at 4 °C to pellet the cells. Carefully remove and discard the supernatant.
    8. Wash the cells by resuspending the cell pellet with ice-cold nuclease-free PBS.
    9. Aliquot the cell suspension to each new microcentrifuge tube.
    10. Centrifuge the cell suspension at 300 x g for 5 min at 4 °C to pellet the cells. Carefully remove and discard the supernatant.
    11. Add 500 μl of lysis buffer (+) to each tube containing the cell pellet, and vortex thoroughly.
    12. Incubate the tube for 10 min at 4 °C or on ice.
    13. Centrifuge the cell suspension at 12,000 x g for 5 min at 4 °C.

  4. Preclear step
    1. Transfer the supernatant (cell lysate) to the tube (prepared in step 14) containing protein A or protein G agarose beads washed once with lysis buffer (+); that were prepared in steps 6-14.
    2. Incubate the tube with rotation for 1 h at 4 °C.

  5. Washing the antibody-immobilized protein A or protein G agarose beads
    During Preclear step, wash the antibody-immobilized protein A or protein G agarose beads once with 1 mL of lysis buffer (+).
    1. Centrifuge the tube (prepared in step 5) containing antibody-immobilized protein A or protein G agarose beads at 2,000 x g for 1 min at 4 °C.
    2. Discard the supernatant carefully.
    3. Add 1 ml of lysis buffer (+), and mix briefly, then centrifuge the tube at 2,000 x g for 1 min at 4 °C.
    4. Discard the supernatant carefully.

  6. Preparation of antibody-immobilized protein A or protein G agarose beads-RNP complex
    1. Centrifuge the tube (prepared in step 29) containing cell lysate and protein A or protein G agarose beads at 2,000 x g for 1 min at 4 °C.
      Notes:
      1. Preparation of Quality Check (QC) sample
        In order to confirm whether RIP-Assay is running properly, we recommend to perform quality check. Collect QC sample and check the protein and RNA expression level at some steps. At least two additional aliquots may be retained for quality check. Use one of the aliquots (10 μl of precleared cell lysate, Input sample) for analysis of RBP expression level by Western Blotting, and use other aliquots (10 μl of precleared cell lysate) for analysis of Total RNA (See Example of RIP-Assay Results).
      2. Preparation of Input sample (for western blotting)
        1. Add 10 μl of Laemmli’s sample buffer to 10 μl of precleared cell lysate, boil for 3-5 min, mix well, and centrifuge.
        2. Resolve 20 μl of the prepared sample on SDS-PAGE, and proceed to western blotting analysis.
      3. Preparation of total RNA (for quality check of Total RNA)
        1. Place 10 μl of precleared cell lysate at -80 °C until beginning of RNA isolation.
        2. After RNP immunoprecipitation, use the lysate to prepare Total RNA sample according to RNA isolation protocol (See below).
    2. Transfer 500 μl of the precleared cell lysate to the tube (prepared in step 33) containing Antibody-immobilized protein A or protein G agarose beads washed once with lysis buffer (+), that was prepared in steps 30-33.
    3. Incubate the tube with rotation for 3 h at 4 °C.

  7. Wash of antibody-immobilized protein A or protein G agarose beads-RNP complex
    1. Centrifuge the tube (prepared in step 36) containing Antibody-immobilized protein A or protein G agarose beads-RNP complex at 2,000 x g for 1 min at 4 °C.
    2. Discard the supernatant carefully.
    3. Add 1 ml of wash buffer (+), mix briefly, and centrifuge the tube at 2,000 x g for 1 min at 4 °C.
    4. Discard the supernatant carefully.
    5. Wash the antibody-immobilized beads-RNP complex twice using steps 39-40.
    6. For fourth wash, add 1 ml of wash buffer (+), then mix well and dispense 100 μl of the mixture to new microcentrifuge tube for QC sample (post-IP beads). Use those aliquots for quality check by western blotting (See Example of RIP-Assay Results).
    7. Resolve 20 μl of the prepared sample on SDS-PAGE, and proceed to western blotting analysis.
    8. Centrifuge the tube containing antibody-immobilized protein A or protein G agarose beads-RNP complex at 2,000 x g for 1 min at 4 °C.
    9. Discard the supernatant carefully.
      Notes:
      1. Preparation of QC sample (for post-IP beads).
      2. Preparation of post-IP beads sample (for western blotting)
        1. Centrifuge the tube containing 100 μl of the mixture at 2,000 x g for 1 min at 4 °C.
        2. Discard the supernatant carefully.
        3. Resuspend the precipitated beads in 20 μl of Laemmli’s sample buffer, boil for 3-5 min, mix well and centrifuge the tube at 2,000 x g for 1 min.

Recipes

  1. Kit components
    1. Lysis buffer
    2. Wash buffer (NT2)
    3. Normal rabbit IgG
    4. High-salt solution
    5. Solution I: Enzyme solution
    6. Solution II: Diluent for solution I
    7. Solution III: Protein dissolvent. Solution III can dissolve protein and dissociate immunocomplex.
    8. Solution IV: Co-precipitator. Solution IV can increase RNA precipitation efficiently.

  2. Commercial reagent
    1. Aprotinin (final concentration 10 μg/ml)
    2. Leupeptin (5 μg/ml)
    3. Phenylmethylsulfonyl fluoride (PMSF) (final concentration 0.5 mM)
    4. RNase inhibitor (Life Technologies, Invitrogen™, catalog number: 10777-019) (final concentration is 50-200 U/ml)
    5. Dithiothreitol (DTT) (reducing agent, final concentration is 1.5 mM)
    6. 100% ethanol (molecular biology grade)
    7. 100% 2-propanol (molecular bio)

  3. Buffer preparation
    1. Lysis buffer
      Add appropriate concentrations of protease inhibitors, RNase inhibitor, and DTT to lysis buffer just before use. Lysis buffer containing these reagents is described as lysis buffer (+) in the following protocols. The optimal concentration of each reagent for RIP-Assay is shown as follows.
    2. Make RSB buffer in DEPEC treated water with final concentrations as follows:
      10 mM Tris (pH 7.5)
      100 mM NaCl
      2.5 mM MgCl2
      Then add other supplements in per ml RSB buffer.
      10 μl digitornin/ml buffer (Digitornin is dissolved in ethanol 4 mg ml-1 freshly), 3 μl RNase inhibitor/ml buffer, 40 μl proteinase inhibitor cocktail/ml.

  4. Wash buffer
    1. Add final 1.5 mM concentration of DTT to wash buffer just before use. Wash buffer containing DTT is described as wash buffer (+) in the following protocols.
    2. Make washing buffer in DEPEC treated water with final concentrations as follows:
      50 mM Tris (pH 7.5)
      50 mM NaCl
      1 mM MgCl2
      0.05% Nonidet P40

  5. Precaution: Additional buffer preparation
    In some cases, both the lysis buffer (+) and wash buffer (+) may require the addition of appropriate volumes of high-salt solution (in these cases, add 30 μl of high-salt solution to each ml of lysis buffer and wash buffer).

  6. RNA isolation (from Antibody-immobilized protein A or protein G agarose beads-RNP complex)
    Solution II and Solution III should be equilibrated to room temperature before use.
    Reagents should be briefly but thoroughly mixed before use.
    1. Prepare master mix solution by diluting 10 μl of Solution I with 390 μl of Solution II per sample.
    2. Dispense 2 μl of Solution IV to each new microcentrifuge tube for step 5.
    3. Add 400μl of Master mix solution to each tube (prepared in RIP-step 44) containing
    4. Antibody-immobilized protein A or protein G agarose beads-RNP complex (obtained in previous).
    5. RNP Immunoprecipitation, vortex thoroughly, then spin-down.
    6. Add 250 μl of Solution III to each tube, vortex thoroughly, then centrifuge the tube at 2,000•x g for 2 min at RT.
    7. Carefully transfer the supernatant to the tube containing 2 μl of Solution IV prepared in step 2 (avoid removing the protein A or protein G agarose beads from the pellet. Contamination of the beads may affect following steps).
    8. Add 600 μl of ice-cold 2-propanol to each tube, vortex briefly but thoroughly, then spin-down.
    9. Incubate the tube at -20 °C or below for 20 min (or for overnight, if necessary).
    10. Centrifuge the tube at 12,000 x g for 10 min at 4 °C, then aspirate the supernatant carefully.
    11. Rinse the pellet with 500 μl of ice-cold 70% ethanol, and mix briefly.
    12. Centrifuge the tube at 12,000 x g for 3 min at 4 °C, then aspirate the supernatant carefully.
    13. Rinse the pellet once again using steps 9-10.
    14. Dry up the pellet by aspirating excess ethanol followed by evaporation for 5-15 min at RT. Avoid RNase contamination (evaporation in clean bench is recommended).
    15. Reconstitute the pellet in 20 μl of nuclease-free water.
    16. Store at -80 °C until starting following analysis.

Acknowledgments

This protocol was adapted from the NBL RIP-Assay Kit (see Reference 1).

References

  1. Protocol from NBL RIP-Assay Kit (catalog number: RN1001).

简介

基因表达的转录后调节是核糖核蛋白(RNP)驱动的过程,其涉及调节剪接,核出口,亚细胞定位,mRNA稳定性和翻译的RNA结合蛋白(RBP)和非编码RNA。编码在特定细胞过程或途径中起作用的蛋白质的mRNA可在由mRNA和RNP组成的独特mRNP复合物内发现。这不仅提供了关于特定过程或途径的已知组分的有价值信息,而且重要的是导致鉴定代表潜在治疗靶标和生物标志物的新组分。除了通过途径扩增鉴定的那些靶标之外,调节RNA功能的特异性RBP(RNA结合蛋白)可以是它们自己的潜在治疗靶标。 RNP-IP是一种允许使用针对RBP的抗体从细胞提取物中分离和鉴定RNP复合物的mRNA,微小RNA和蛋白质组分的技术。一旦纯化,使用各种分子生物学工具例如RT-PCR,基于微阵列技术的基因表达分析(芯片分析)或测序,分析复合物中存在的RNA以鉴定靶mRNA。使用该方法,可以获得存在于细胞核中的更多的RNA。

材料和试剂

  1. 正常兔IgG
  2. 高盐溶液
  3. RIP认证的抗体(NBL,目录号取决于您要定位的目标)
  4. 蛋白酶抑制剂(Molecular Biology)
  5. 抑肽酶
  6. 亮肽素
  7. 苯甲基磺酰氟(PMSF)
  8. RNase抑制剂(Life Technologies,Invitrogen TM,目录号:10777-019)
  9. 二硫苏糖醇(DTT)(还原剂)
  10. 蛋白A珠(GE Healthcare Dharmacon,目录号:17-0780-01)或Protein G珠(Thermo Fisher Scientific,目录号:22852)
  11. 乙醇(分子生物学)
  12. 2-丙醇(分子生物学)
  13. 无核酸酶的PBS
  14. 同种型对照IgG(如有必要)
  15. 套件组件(参见配方)
  16. 商业试剂(见配方)
  17. 缓冲液准备(参见配方)
  18. 洗涤缓冲液(见配方)
  19. 注意:附加缓冲液准备(参见配方)
  20. 抗体固定化蛋白A或蛋白G琼脂糖珠-RNA复合物的制备

设备

  1. 微量离心机能够处理15,000个电子邮件
  2. 微量离心管(1.5ml或2ml)(无核酸酶)(建议;使用低附着管RIP-测定)
  3. 离心机能够2,000 x g
  4. 离心管(15ml或50ml)
  5. 移液管(5ml,10ml,25ml)(不含核酸酶)
  6. 移液管吸头(10μl,20-100μl,200μl和1,000μl)(不含核酸酶)(推荐使用RIP-Assay的低附着力吸头)
  7. 超低温冰箱(-80℃)
  8. 冰柜(-20°C以下)
  9. 端到端旋转器
  10. 涡流搅拌器
  11. 手套

程序

  1. 前处理:制备抗体固定化蛋白A或蛋白G琼脂糖珠。
    1. 用等量的不含核酸酶的PBS洗涤蛋白A或蛋白G琼脂糖珠3次(在4℃下离心2 000×g 1分钟)。
    2. 等分25μl的50%珠浆液到每个新的微量离心管。
    3. 每管加入1 ml洗涤缓冲液(+)
    4. 向每个管中加入15μg抗体(分别为正常兔IgG作为阴性对照或针对目标RBP的RIP-认证抗体)。
    5. 孵育管在4℃下旋转至少30分钟。 如果需要,可以将该温育延长至过夜。

  2. 前步:制备用于预澄清的蛋白A或蛋白G琼脂糖珠
    1. 用等量的不含核酸酶的PBS洗涤蛋白A或蛋白G琼脂糖珠3次(在4℃下离心2 000×g 1分钟)。
    2. 等分25μl的50%珠浆液到每个新的微量离心管
    3. 向每个管中加入500μl洗涤缓冲液(+),并短暂混合。
    4. 在4℃下以2,000×g离心管1分钟。
    5. 小心弃去上清液。
    6. 将珠子留在4°C或冰上,直到开始预清洗步骤
    7. 在Preclear步骤前,用500μl裂解缓冲液(+)洗涤珠一次
    8. 在4℃下,以2,000×g离心管1分钟。
    9. 小心弃去上清液。 使用这些蛋白A或蛋白G琼脂糖珠,用裂解缓冲液(+)洗涤一次用于预澄清步骤(步骤28)。

  3. 哺乳动物细胞溶解
    注意:为了获得"高质量RNA",应在RIP-Assay中使用新鲜培养的细胞。
    1. 如果需要,通过吸液或使用细胞刮刀从培养皿中分离细胞。 收集细胞悬浮液在离心管中。
    2. 在4℃下将细胞悬浮液以300×g离心5分钟以沉淀细胞。 小心取出并弃去上清液。
    3. 通过用冰冷的PBS重悬细胞沉淀来清洗细胞
    4. 在4℃下将细胞悬浮液以300×g离心5分钟以沉淀细胞。 小心取出并弃去上清液。
    5. 使用步骤17-18再次洗涤细胞。
    6. 通过用冰冷的无核酸酶的PBS重悬细胞沉淀来洗涤细胞。
    7. 在4℃下将细胞悬浮液以300×g离心5分钟以沉淀细胞。 小心取出并弃去上清液。
    8. 通过用冰冷的无核酸酶的PBS重悬细胞沉淀来清洗细胞
    9. 将细胞悬浮液分装到每个新的微量离心管中
    10. 在4℃下将细胞悬浮液以300×g离心5分钟以沉淀细胞。 小心取出并弃去上清液。
    11. 向每个含有细胞沉淀的管中加入500μl裂解缓冲液(+),彻底涡旋。
    12. 在4℃或冰上孵育管10分钟。
    13. 在4℃下,将细胞悬浮液以12,000×g离心5分钟

  4. 预清晰步骤
    1. 将上清液(细胞裂解物)转移至含有蛋白A或蛋白G琼脂糖珠的试管(在步骤14中制备),用裂解缓冲液(+)洗涤一次; 在步骤6-14中制备。
    2. 孵育管在4℃下旋转1小时。

  5. 洗涤抗体固定的蛋白A或蛋白G琼脂糖珠
    在预处理步骤中,用1 mL裂解缓冲液(+)洗涤一次抗体固定的蛋白A或蛋白G琼脂糖珠。
    1. 在4℃下,将含有抗体固定的蛋白A或蛋白G琼脂糖珠的管(在步骤5中制备)在2,000×g离心1分钟。
    2. 小心弃去上清液。
    3. 加入1ml裂解缓冲液(+),并短暂混合,然后在4℃下以2,000×g离心该管1分钟。
    4. 小心弃去上清液。

  6. 抗体固定化蛋白A或蛋白G琼脂糖珠-RNA复合物的制备
    1. 在4℃下将含有细胞裂解物和蛋白A或蛋白G琼脂糖珠的管(在步骤29中制备)以2,000xg离心1分钟。
      注意:
      1. 准备质量检查(QC)示例
        为了确认RIP-Assay是否正常运行,我们建议执行质量检查。收集QC样品,并检查蛋白质和RNA表达水平在一些步骤。可以保留至少两个另外的等分试样用于质量检查。使用一个等分试样(10μl预清除的细胞裂解物,输入样品)通过蛋白质印迹分析RBP表达水平,并使用其他等分试样(10μl预清除的细胞裂解物)分析总RNA(参见RIP-测定的实施例结果)。
      2. 输入样品的制备(用于western印迹)
        1. 将10μlLaemmli样品缓冲液加入10μl预清除的细胞裂解物中,煮沸3-5分钟,充分混合,离心。
        2. 在SDS-PAGE上分离20μl制备的样品,并进行western印迹分析。
      3. 总RNA的制备(用于总RNA的质量检查)
        1. 将10μl预清除的细胞裂解物置于-80℃下,直到开始RNA分离。
        2. 在RNP免疫沉淀后,使用裂解物根据RNA分离方案制备总RNA样品(见下文)。
    2. 将500μl预清除的细胞裂解物转移到含有抗体固定的蛋白A或蛋白G琼脂糖珠的试管(在步骤33中制备),其在步骤30-33中制备的裂解缓冲液(+)洗涤一次。
    3. 孵育管在4℃下旋转3小时。

  7. 洗涤抗体固定蛋白A或蛋白G琼脂糖珠-RNP复合物
    1. 在4℃下将含有抗体固定化蛋白A或蛋白G琼脂糖珠-RNA复合物的管(在步骤36中制备)以2,000×g离心1分钟。
    2. 小心弃去上清液。
    3. 加入1ml洗涤缓冲液(+),短暂混合,并在2,000×g离心管在4℃下1分钟。
    4. 小心弃去上清液。
    5. 使用步骤39-40洗涤抗体固定珠-RNP复合物两次。
    6. 对于第四次洗涤,加入1ml洗涤缓冲液(+),然后充分混合并将100μl混合物分配到新的微量离心管的QC样品(post-IP珠)。 使用这些等分试样通过蛋白质印迹进行质量检查(参见RIP-测定结果的实施例)。
    7. 在SDS-PAGE上分离20μl制备的样品,并进行western印迹分析。
    8. 在4℃下,将含有抗体固定的蛋白A或蛋白G琼脂糖珠-RNA复合物的管在2,000×g离心1分钟。
    9. 小心弃去上清液。
      注意:
      1. 准备QC样品(用于后IP珠)。
      2. 后IP珠粒样品的制备(用于western印迹)
        1. 在4℃下,将含有100μl混合物的管在2,000×g离心1分钟。
        2. 仔细弃去上清液。
        3. 将沉淀的珠子重悬在20μlLaemmli的样品缓冲液中,煮沸3-5分钟,充分混合,并以2,000×g离心1分钟。

食谱

  1. 套件组件
    1. 裂解缓冲液
    2. 洗涤缓冲液(NT2)
    3. 正常兔IgG
    4. 高盐溶液
    5. 溶液I:酶溶液
    6. 溶液II:溶液I的稀释剂
    7. 溶液III:蛋白质溶解。 溶液III可以溶解蛋白质和解离免疫复合物
    8. 溶液IV:共沉淀器。 溶液IV可以有效地增加RNA沉淀

  2. 商业试剂
    1. 抑肽酶(终浓度10μg/ml)
    2. 亮肽素(5μg/ml)
    3. 苯甲基磺酰氟(PMSF)(终浓度0.5mM)
    4. RNase抑制剂(Life Technologies,Invitrogen TM,目录号:10777-019)(终浓度为50-200U/ml)
    5. 二硫苏糖醇(DTT)(还原剂,终浓度为1.5mM)
    6. 100%乙醇(分子生物学级)
    7. 100%2-丙醇(分子生物)

  3. 缓冲液制备
    1. 裂解缓冲液
      在使用前,向裂解缓冲液中加入适当浓度的蛋白酶抑制剂,RNA酶抑制剂和DTT。 含有这些试剂的裂解缓冲液在以下方案中被描述为裂解缓冲液(+)。 用于RIP-测定的每种试剂的最佳浓度如下所示
    2. 在DEPEC处理水中制备RSB缓冲液,最终浓度如下:
      10mM Tris(pH7.5) 100 mM NaCl
      2.5mM MgCl 2 v/v 然后在每ml RSB缓冲液中加入其他补充剂。
      10μldigitornin/ml缓冲液(Digitornin溶于乙醇4mg ml-新鲜),3μlRNA酶抑制剂/ml缓冲液,40μl蛋白酶抑制剂混合物/ml。

  4. 洗涤缓冲液
    1. 恰好在使用前向洗涤缓冲液中加入最终1.5mM DTT。 在以下方案中,含有DTT的洗涤缓冲液被描述为洗涤缓冲液(+)。
    2. 在DEPEC处理水中制备洗涤缓冲液,最终浓度如下:
      50mM Tris(pH7.5) 50mM NaCl 1mM MgCl 2
      0.05%Nonidet P40

  5. 注意:附加缓冲液准备
    在一些情况下,裂解缓冲液(+)和洗涤缓冲液(+)可能需要加入适当体积的高盐溶液(在这些情况下,向每ml裂解缓冲液中加入30μl高盐溶液, 缓冲)。

  6. RNA分离(来自抗体固定的蛋白A或蛋白G琼脂糖珠-RNA复合物)
    在使用之前,溶液II和溶液III应当平衡至室温。
    1. 通过用每个样品390μl的溶液II稀释10μl溶液I制备主混合溶液
    2. 向每个新的微量离心管中加入2μl溶液IV用于第5步
    3. 向含有
      的每管(在RIP-步骤44中制备)中加入400μl主混合物溶液
    4. 抗体固定的蛋白A或蛋白G琼脂糖珠-RNA复合物(在前面获得)。
    5. RNP免疫沉淀,彻底涡旋,然后离心
    6. 向每个管中加入250μl溶液III,彻底涡旋,然后在室温下以2,000×g×g离心2分钟。
    7. 小心地将上清液转移到含有2μl在步骤2中制备的溶液IV的管(避免从沉淀物中去除蛋白A或蛋白G琼脂糖珠),珠的污染可能影响以下步骤)。
    8. 向每个管中加入600μl冰冷的2-丙醇,短暂地但短暂地涡旋,然后旋转。
    9. 孵育管在-20°C或以下20分钟(或过夜,如果必要)。
    10. 在4℃下以12,000×g离心管10分钟,然后小心地抽吸上清液。
    11. 用500μl冰冷的70%乙醇冲洗沉淀,并短暂混合
    12. 在4℃下以12,000xg离心管3分钟,然后小心地抽吸上清液。
    13. 使用步骤9-10再次冲洗沉淀。
    14. 通过吸出过量的乙醇,然后在室温下蒸发5-15分钟来干燥沉淀。 避免RNase污染(建议在清洁工作台中蒸发)。
    15. 用20μl无核酸酶的水重建沉淀。
    16. 储存于-80°C,直到开始分析之后。

致谢

该协议改编自NBL RIP-测定试剂盒(参见参考文献1)。

参考文献

  1. 来自NBL RIP-Assay Kit(目录号:RN1001)的方案。
  • English
  • 中文翻译
免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2012 The Authors; exclusive licensee Bio-protocol LLC.
引用:Liu, F. (2012). RNP-IP (Original protocol)-getting majority of RNA from RNA binding protein in nucleus. Bio-protocol Bio101: e218. DOI: 10.21769/BioProtoc.218;
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Shreyas Jadhav
Brigham And Women's Hospital
Could I use the same protocol for RIP from Tissue Lysates?

Thanks,

Shreyas
9/29/2014 9:10:45 AM Reply
FengZhi Liu
School of Biomedical Sciences, Thomas Jefferson University, USA

Hi,

I only used cell lysates to get the target protein. You know the principle of this method is to use specificity of Ag-Ab reaction. If your target protein is outside of the cells, you should think about it amount is enough or not for this method. From theory, it is feasible. the problem is how do you extract the raw protein products. If you use other biochemical methods to purify the target protein , then run RIP, it may be better.

9/29/2014 1:47:33 PM


城 王
中科院
3/5/2012 10:30:02 AM Reply
FengZhi Liu
School of Biomedical Sciences, Thomas Jefferson University, USA

Here is the link:
https://ruo.mbl.co.jp/gtf/1/1/RN1001.pdf

3/7/2012 5:43:07 AM