Determining Ribosome Translational Status by Ribo-ELISA
采用Ribo-ELISA检测核糖体的翻译状态   

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参见作者原研究论文

本实验方案简略版
The Journal of Cell Biology
Apr 2012

 

Abstract

The Ribo-ELISA was originally developed to elucidate the basis for the ribopuromycylation method (RPM)-based detection of ribosome bound nascent chains. The Ribo-ELISA enables characterization of the translational status of ribosomes, and can be applied to the discovery of super-ribosomal complexes with novel ribosome associated macromolecules that are isolated by physical fractionation in sucrose gradients or other methods.

Keywords: Ribosome (核糖体), ELISA (ELISA), Ribopuromycylation (Ribopuromycylation)

Background

Ribosomes are heterogeneous structures consisting of 40S and 60S subunits that are present in cells as monosomes and polysomes, when multiple ribosomes are bound to a single mRNA. Additionally, translating ribosomes can be associated with multiple molecular complexes that modulate translation. The ribosome ELISA (Enzyme-Linked ImmunoSorbent Assay) enables detection of translating ribosomes by in vitro puromycylation of ribosome associated nascent chains (David et al., 2012). This chemical reaction proceeds spontaneously upon adding puromycin to ribosomes with bound nascent chains. This method quantitates the number of nascent chains present per ribosome and can be used to determine the translational status of monosomes relative to polysomes, and identify ribosomes bound to other macromolecules that alter its sedimentation rate or migration in sizing columns.

Materials and Reagents

  1. Materials
    1. 175 cm2 flasks (Corning, catalog number: 431306 )
    2. Thinwall 13.2 ml polypropylene tube (Beckman Coulter, catalog number: 331372 )
    3. Parafilm (Bemis, catalog number: 701606 )
    4. Pipette tips (MµltiGuardTM Tips 1-100 µl, 1-200 µl, 100-1,000 µl) (Sorenson BioScience, catalog numbers: 36060 , 14220 , 14200 )
    5. 1.5 ml Eppendorf tubes (Sigma-Aldrich, catalog number: Z666505 )
      Manufacturer: Eppendorf, catalog number: 022431081 .
    6. 96-well Millipore Multiscreen HTS PVDF plates (Merck, catalog number: MSIPS4W10 )
    7. Paper towels

  2. Cell line(s)
    Though this procedure was originally designed for HeLa cells, it can be easily adapted for any other cell lines. The described procedure and associated picture (Figure 1) uses HeLa cells (ATCC, catalog number: CCL-2.1 )

  3. Reagents
    1. Trypsin-EDTA (Thermo Fisher Scientific, GibcoTM, catalog number: 25300054 )
    2. StartingBlockTM blocking buffer (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 37538 )
    3. Hydrogen chloride (HCl) 1.6%
    4. Potassium phosphate monobasic (KH2PO4)
    5. Sodium chloride (NaCl)
    6. Na2HPO4·7H2O (Life Technologies)
    7. Dulbecco’s modified Eagle medium (DMEM) (Thermo Fisher Scientific, GibcoTM, catalog number: 41966029 )
    8. Penicillin/streptomycin (Thermo Fisher Scientific, GibcoTM, catalog number: 10378016 )
    9. Fetal bovine serum (FBS) (Eurobio, catalog number: CVFSVF0101 )
    10. Tris-HCl pH 7.5 (Thermo Fisher Scientific, InvitrogenTM, catalog number: 15567027 )
    11. Magnesium chloride hexahydrate (MgCl2·6H2O) (Sigma-Aldrich, catalog number: M2670 )
    12. Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P9541 )
    13. Sucrose (Sigma-Aldrich, catalog number: 84097 )
    14. NP-40 10% (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 28324 )
    15. Complete Mini EDTA-free protease inhibitor tablets (Sigma-Aldrich, Roche Dignostics, catalog number: 11836170001 )
    16. RNase Out (Life Technologies, catalog number: 100000840 or Thermo Fisher Scientific, InvitrogenTM, catalog number: 10777019 )
    17. DEPC treated water (Thermo Fisher Scientific, InvitrogenTM, catalog number: 750023 )
    18. 16% paraformaldehyde (Electron Microscopy Sciences, catalog number: 15710 )
    19. Glutamine (Thermo Fisher Scientific, GibcoTM, catalog number: 25030081 )
    20. 70% ethanol (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: R40135 )

  4. Protein synthesis inhibitors
    1. Cycloheximide (CHX) (Sigma-Aldrich, catalog number: C7698 )
      Stock solution (1,000x): 100 mg/ml (or 355 mM) in 50% ethanol
      Store at -20 °C
    2. Emetine dihydrochloride (Emetine) (Sigma-Aldrich, catalog number: E2375 )
      Stock solution (1,000x): 25 mg/ml (or 45 mM) in 50% ethanol
      Store at -20 °C
    3. Puromycin (PMY) (Sigma-Aldrich, catalog number: P7255 )
      Stock solution (1,000x): 50 mg/ml (or 91 mM) in 50% ethanol
      Store at -20 °C

  5. Antibodies
    1. Primary antibodies
      1. Anti-puromycin mouse monoclonal antibodies: hybridoma clone 2A4 is available to the scientific community from the Developmental Studies Hybridoma Bank http://dshb.biology.uiowa.edu/PMY-2A4
      2. Anti-ribosomal P antibody: human polyclonal autoimmune antiserum (from lupus patients) which recognizes three proteins of the 60S ribosomal subunit, RPLP0, P1 and P2 (ImmunoVision, catalog number: HPO-0300 )
    2. Secondary antibodies
      1. Rabbit anti-mouse HRP conjugate (Cappel) (MP Biomedical, catalog number: 55564 )
      2. Goat anti-human Ig-HRP (Jackson ImmunoResearch, catalog number: 109-035-003 )

  6. Solutions (see Recipes)
    1. Phosphate buffered saline (PBS)
    2. Growth medium
    3. Homogenization buffer
    4. Polysome buffer
    5. 15% sucrose solution
    6. 50% sucrose solution
    7. 3% PFA

Equipment

  1. PIPETMAN 1-20 µl (Sartorius, model: Proline® Plus, catalog number: 728030 )
  2. PIPETMAN 20-200 µl (Sartorius, model: Proline® Plus, catalog number: 728060 )
  3. PIPETMAN 100-1,000 µl (Sartorius, model: Proline® Plus, catalog number: 728070 )
  4. Water bath (JULABO, model: TW20 )
  5. Class II laminar flow hood (FASTER, model: SafeFAST Elite )
  6. Cell Incubator (Heraeus, model: HERACell )
  7. Centrifuge (Eppendorf, model: 5415 R )
  8. Inverted microscope (Nikon Instruments, model: Eclipse TS100 )
  9. Microplate photometer (TECAN, model: Infinite M200 )
  10. Dounce homogenizer (DWK Life Sciences, Kimble, catalog number: 885300-0002 )
  11. Ceramic bead tubes Lysing matrix D (MP Biomedicals, catalog number: 116913050 )
  12. FastPrep-24 MP device (MP Biomedicals, catalog number: 116004500 )
  13. Ultracentrifuge (Beckman Coulter)
  14. Rotor (Beckman Coulter, model: SW 41 Ti )
  15. MultiScreenHTS Vacuum manifold (Merck, catalog number: MSVMHTS00 )
  16. Optional: Density gradient fractionator (Teledyne Isco)

Software

  1. GraphPad Prism software

Procedure

The following procedure describes the first application of Ribo-ELISA (David et al., 2012) which was used to detect puromycin association with ribosomes: at that time it was not clear whether puromycin binding was limited to translating ribosomes.
Either emetine or CHX can be used as elongation inhibitors to freeze polysome. Unlike CHX, emetine irreversibly inhibits translation, and need not be maintained throughout the procedure.
Day 1:

  1. Pre-warm growth medium (see Recipes), PBS and trypsin-EDTA in 37 °C water bath. Trypsinize two 175 cm2 flasks of cells at 70%-80% confluency. Remove the medium, wash with 10 ml of warm PBS, add 5 ml of trypsin-EDTA and leave for 5 min at 37 °C. Resuspend cells with 5 ml of growth medium.
  2. Seed two 175 cm2 flasks with 1 x 107 HeLa cells per flask (Corning) containing 15 ml of warm growth medium each. Incubate for 30 h to allow HeLa cells to attach and proliferate.
  3. Sucrose gradients (see Video 1). Prepare 15% and 50% sucrose solutions (see Recipes). Add 5 ml of 50% sucrose solution in 13.2 ml Polypropylene centrifuge tube (Beckman Coulter). Tilt the tube (almost 90°) and carefully add 5 ml of 15% sucrose solution on the top (add slowly the solution flowing along the tube wall and slowly put the tube back to vertical). Seal the tube with Parafilm and gently put the tube in horizontal position on the bench. Wait 1-2 h and put it back to the vertical. The 15-50% sucrose gradient is ready. Store it in the fridge O.N.
    Note: Other methods can be used to prepare gradients. A convenient alternative is the freeze-thawing method which entails successive freezing (-80 °C) of 2 ml layered sucrose solutions starting from the highest sucrose concentration (50%, 40%, 30%, 20%, 10% w/v sucrose solutions) (Bastide et al., 2017).

    Video 1. Sucrose gradients preparation. This video provides step by step guidelines for preparing 15% and 50% sucrose gradients.

Day 2:

  1. Examine HeLa cells under an inverted microscope to ensure right confluence (between 60-70%).
  2. Incubate cells for 5 min with 10 ml of warm growth medium supplemented with 100 µg/ml CHX (or 20 µg/ml emetine) added directly to the medium.
  3. Remove the medium, wash twice with 10 ml of warm PBS. Add 5 ml of trypsin-EDTA and put the cells back in the incubator for 5 min, then resuspend cells with 5 ml of growth medium, centrifuge at 400 x g for 5 min and wash the pellet twice with 10 ml of ice-cold PBS (400 x g/5 min/4 °C) (both solutions supplemented with 100 µg/ml CHX, see Recipes).
    Note: From this step RNase free tips should be used.
  4. Pellet cells by centrifugation (400 x g/5 min/4 °C). Resuspend cells in 1 ml ice-cold homogenization buffer (see Recipes).
  5. Transfer the cell suspension to ceramic beads tubes (Lysing matrix D) on ice.
    Note: Importantly, NP-40 in homogenization buffer is not essential when using ceramic beads to lyse cells: ribosomes are recovered with similar efficiency. Therefore, Ribosome ELISA can be performed in the absence of detergent.
  6. ‘Vortex’ at RT for 20 sec (using the FastPrep-24 MP device).
    Note: In lieu of ceramic beads, cells can be stroked 10 times in ice cold homogenization buffer using Dounce homogenizer embedded in ice.
  7. Transfer supernatant to 1.5 ml Eppendorf tubes. Spin at 13,000 x g for 10 min at 4 °C.
  8. Carefully collect the supernatant.
    Note: If the supernatant is not perfectly clear, recentrifuge.
  9. Slowly layer 1 ml clear lysate along the tube wall on the top of the cold 15-50% sucrose gradient. Balance the rotor with another sample or a tube filled with water. Spin at 151,263 x g (35,000 rpm) (Beckman Coulter, rotor SW41.Ti) for 2.5 h at 4 °C.
  10. Fractionate (1 ml fractions) and measure absorbance continuously at 254 nm using a gradient system automate (Teledyne Isco). You will get 11 fractions.
    Note: If you lack access to an automated gradient collector, fractions can be collected in the old-fashioned way; manually from the top. Use a PIPETMAN 100-1,000 µl with a 1 ml tip. Push the piston to the 1 ml max, and place the tip in contact with the meniscus. Carefully, collect 1 ml sucrose by releasing the pipette piston slowly. Always keep the tip in contact with the meniscus. Put each fraction in a clean 1.5 ml tube (11 fractions).
  11. Add 100 µl of each fraction to wells of a 96-well Millipore Multiscreen HTS PVDF plate (11 wells, lane 1). Repeat this operation five times in following lanes to get technical replicates (3 wells per fraction) for two experimental conditions (anti-puromycin staining and anti-P protein staining). Load an identical plate as control (‘no puromycin labeling’). Incubate plates for 10 min on ice, and then vacuum the liquid through using the MultiScreenHTS Vacuum manifold.
  12. Add 200 µl/well of polysome buffer (see Recipes) and vacuum again.
  13. Puromycin labeling. Add 100 µl of PMY (91 µM) containing polysome buffer to the experimental plate and 100 µl of polysome buffer to the control plate. Incubate for 10 min on ice. In parallel, skip this step in order to generate the ‘no puromycin labeling’ control plate.
  14. Wash. Flick plate out into sink, briefly drain inverted on paper towels. Add 200 µl of polysome buffer, flick plate out again. Repeat this operation twice.
  15. Fixation. Add 100 µl of 3% paraformaldehyde (PFA) (see Recipes) inside the hood. Incubate for 10 min at room temperature (RT). Dispose of the PFA waste properly.
    Note: We used this fixation step in the original protocol to mimic the RPM procedure on cells. However, this step is not necessary to detect puromycylated nascent chains.
  16. Wash. Wash wells with 200 µl PBS 3 times.
  17. Membrane blocking. Incubate with 100 µl of StartingBlockTM blocking buffer for 30 min at RT. Vacuum.
    Note: In the original procedure, StartingBlockTM gave very low background Ab binding. Using different Abs, other blocking buffers might be superior.
  18. Primary antibody. Incubate for 1 h with 50 µl of either 2.5 µg/ml anti-PMY mAb (in StartingBlockTM) at RT or anti-Ribosomal P antibodies (1/5,000, in StartingBlockTM)
  19. Wash. Wash wells with 200 µl PBS 3 times.
  20. Secondary antibody. Incubate with 50 µl of secondary antibody HRP conjugate, rabbit anti-mouse for PMY staining and goat anti-human for ribosomal P staining (1/100 in StartingBlockTM) for 1 h at RT.
  21. Wash. Wash wells with 200 µl PBS 3 times.
  22. Signal Generation. Add 100 µl of TMB substrate (KPL) to each well. When some blue color becomes visible in the well, stop the reaction using 50 µl of 1.6% HCl. Transfer the liquid in each well to a plastic plate with a transparent bottom. Then, measure absorbance at 450 nm.
  23. Quantification. Determine the level of background staining by using the average of unlabeled wells from the control plate and subtract it from test values. In the illustrated example, data were graphed using GraphPad Prism software (Figure 1).


    Figure 1. Schematic representation of Ribosome ELISA applied to detect PMY association with translating ribosomes (from David et al., 2012). HeLa cells or HeLa cells incubated with emetine for 15 min were lysed and fractionated on 15-50% sucrose gradients. Fractions were bound to PVDF 96-well plates and incubated with PMY, which results in ribosome-catalyzed puromycylation. Ribosomes were detected by A260 of fractions or by ELISA for the ribosomal P proteins (here resolved into the 3 known species) as detected by human autoimmune Abs, which establishes that 60S subunit and monosome (80S) were bound to PVDF (fractions 4 and 5). Puromycylation was detected by ELISA for PMY using 12-D10, and clearly demonstrates that monosomes and free 60S subunits do not stably associate with PMY, which requires nascent chains.

Data analysis

For data analysis, we previously used GraphPad Prism software. An example is presented in the following paper: David et al., 2012.

Notes

Polysome profile can vary dramatically depending on the cell line and translation rate.

Recipes

  1. Phosphate buffered saline (PBS)
    210.0 mg/L KH2PO4
    9,000 mg/L NaCl
    726.0 mg/L Na2HPO4·7H2O (Life Technologies)
  2. Growth medium
    Dulbecco’s modified Eagle’s medium with glutamine (Thermo Fisher Scientific)
    1% penicillin/streptomycin
    7.5% fetal bovine serum (FBS)
  3. Homogenization buffer
    50 mM Tris-HCl pH7.5
    5 mM MgCl2
    25 mM KCl
    0.2 M sucrose (Sigma-Aldrich)
    0.5% NP-40 (Thermo Fisher Scientific)
    100 µg/ml CHX (Sigma-Aldrich)
    EDTA-free protease inhibitors (Roche)
    10 U/ml RNase Out (Life technologies)
    DEPC water (Thermo Fisher Scientific)
  4. Polysome buffer
    50 mM Tris-HCl pH 7.5
    5 mM MgCl2
    25 mM KCl
    0.2 M sucrose (Sigma-Aldrich)
    100 µg/ml CHX (Sigma-Aldrich)
    EDTA-free protease inhibitors (Roche)
    10 U/ml RNase Out (Life technologies)
    DEPC water (Thermo Fisher Scientific)
  5. 15% sucrose solution
    15% sucrose (m/v)
    50 mM Tris-HCl pH 7.5
    5 mM MgCl2
    25 mM KCl
    100 µg/ml CHX (Sigma-Aldrich)
    EDTA-free protease inhibitors (Roche)
    10 U/ml RNase Out (Life technologies)
    DEPC water (Thermo Fisher Scientific)
  6. 50% sucrose solution
    50% sucrose (m/v)
    50 mM Tris-HCl pH 7.5
    5 mM MgCl2
    25 mM KCl
    100 µg/ml CHX (Sigma-Aldrich)
    EDTA-free protease inhibitors (Roche)
    10 U/ml RNase Out (Life technologies)
    DEPC water (Thermo Fisher Scientific)
  7. 3% PFA
    Dilute stock solution (16%) in PBS 1x

Acknowledgments

JWY is supported by the Division of Intramural Research, National Institute of Allergy and Infectious Diseases. AD benefits from generous funding from Fondation pour la Recherche Médicale, Ligue contre le Cancer and Cancéropôle GSO. The authors declare no conflicts of interest or competing interests.

References

  1. Bastide, A., Peretti, D., Knight, J. R., Grosso, S., Spriggs, R. V., Pichon, X. Sbarrato, T., Roobol, A., Roobol, J., Vito, D., Bushell, M., von der Haar, T., Smales, C. M., Mallucci, G. R. and Willis, A. E. (2017). RTN3 is a novel cold-Induced protein and mediates neuroprotective effects of RBM3. Curr Biol CB 27: 638-650.
  2. David, A., Dolan, B. P., Hickman, H. D., Knowlton, J. J., Clavarino, G., Pierre, P., Bennink, J. R. and Yewdell, J. W. (2012). Nuclear translation visualized by ribosome-bound nascent chain puromycylation. J Cell Biol 197(1): 45-57.

简介

Ribo-ELISA最初是为阐明基于核糖核苷酸化方法(RPM)的核糖体结合新生链检测的基础而开发的。 Ribo-ELISA能够表征核糖体的翻译状态,并且可以应用于通过蔗糖梯度中的物理分离或其他方法分离的新型核糖体相关大分子的超核糖体复合物的发现。

【背景】核糖体是由40S和60S亚单位组成的异构结构,当多个核糖体与单个mRNA结合时,它们以单体和多核糖体存在于细胞中。 另外,翻译核糖体可以与调节翻译的多个分子复合物相关联。 核糖体ELISA(Enzyme-Linked ImmunoSorbent Assay)能够通过核糖体相关新生链的体外嘌呤化检测翻译核糖体(David等人,2012)。 在将嘌呤霉素添加至具有结合的新生链的核糖体时,这种化学反应自发进行。 该方法定量测定每个核糖体中出现的新生链的数量,并且可以用于确定单核细胞相对于多核糖体的翻译状态,并鉴定结合其他大分子的核糖体,其改变其在沉淀柱中的沉降速率或迁移。

关键字:核糖体, ELISA, Ribopuromycylation

材料和试剂

  1. 物料
    1. (Corning,目录号:431306)
    2. 薄壁13.2毫升聚丙烯管(Beckman Coulter,目录号:331372)
    3. Parafilm(Bemis,产品目录号:701606)
    4. 移液器吸头(MμltiGuardTM吸头1-100μl,1-200μl,100-1000μl)(Sorenson BioScience,目录号:36060,14220,14200)
    5. 1.5ml Eppendorf管(Sigma-Aldrich,目录号:Z666505)
      制造商:Eppendorf,目录号:022431081。
    6. 96孔Millipore Multiscreen HTS PVDF板(Merck,目录号:MSIPS4W10)
    7. 纸巾

  2. 细胞系(S)
    虽然这个程序最初是为HeLa细胞设计的,但它可以很容易地适用于任何其他细胞系。所描述的程序和相关图片(图1)使用HeLa细胞(ATCC,目录号:CCL-2.1)。

  3. 试剂
    1. 胰蛋白酶-EDTA(Thermo Fisher Scientific,Gibco TM,目录号:25300054)
    2. StartingBlock TM封闭缓冲液(Thermo Fisher Scientific,Thermo Scientific TM,产品目录号:37538)
    3. 氯化氢(HCl)1.6%
    4. 磷酸二氢钾(KH 2 PO 4)
    5. 氯化钠(NaCl)
    6. Na 2 HPO 4•7H 2 O(Life Technologies)
    7. Dulbecco's改良的Eagle培养基(DMEM)(Thermo Fisher Scientific,Gibco TM,目录号:41966029)
    8. 青霉素/链霉素(Thermo Fisher Scientific,Gibco TM,目录号:10378016)
    9. 胎牛血清(FBS)(Eurobio,目录号:CVFSVF0101)
    10. Tris-HCl pH 7.5(Thermo Fisher Scientific,Invitrogen TM,目录号:15567027)
    11. 氯化镁六水合物(MgCl 2•6H 2 O)(Sigma-Aldrich,目录号:M2670)
    12. 氯化钾(KCl)(Sigma-Aldrich,目录号:P9541)
    13. 蔗糖(Sigma-Aldrich,目录号:84097)
    14. NP-40 10%(Thermo Fisher Scientific,Thermo Scientific TM,目录号:28324)
    15. 完整的无EDTA无蛋白酶抑制剂片剂(Sigma-Aldrich,Roche Dignostics,目录号:11836170001)
    16. RNase Out(Life Technologies,目录号:100000840或Thermo Fisher Scientific,Invitrogen TM,产品目录号:10777019)
    17. DEPC处理的水(Thermo Fisher Scientific,Invitrogen TM,目录号:750023)
    18. 16%多聚甲醛(Electron Microscopy Sciences,目录号:15710)
    19. 谷氨酰胺(Thermo Fisher Scientific,Gibco TM,目录号:25030081)
    20. 70%乙醇(Thermo Fisher Scientific,Thermo Scientific TM,目录号:R40135)

  4. 蛋白质合成抑制剂
    1. 环己酰亚胺(CHX)(Sigma-Aldrich,目录号:C7698)
      储备溶液(1,000x):在50%乙醇中100mg / ml(或355mM)
      在-20°C储存
    2. 依地酸二盐酸盐(Emetine)(Sigma-Aldrich,目录号:E2375)
      储备溶液(1,000x):在50%乙醇中25mg / ml(或45mM)
      在-20°C储存
    3. 嘌呤霉素(PMY)(Sigma-Aldrich,目录号:P7255)
      储备液(1,000x):在50%乙醇中50mg / ml(或91mM)
      在-20°C储存

  5. 抗体
    1. 一抗
      1. 抗嘌呤霉素小鼠单克隆抗体:杂交瘤克隆2A4可从发育研究杂交瘤银行获得科学界 http ://dshb.biology.uiowa.edu/PMY-2A4
      2. 抗核糖体P抗体:识别60S核糖体亚基RPLP0,P1和P2(ImmunoVision,目录号:HPO-0300)的三种蛋白质的人多克隆自身免疫抗血清(来自狼疮患者)
    2. 二抗
      1. 兔抗小鼠HRP结合物(Cappel)(MP Biomedical,目录号:55564)
      2. 山羊抗人Ig-HRP(Jackson ImmunoResearch,目录号:109-035-003)

  6. 解决方案(请参阅食谱)
    1. 磷酸盐缓冲盐水(PBS)
    2. 生长介质
    3. 匀浆缓冲液
    4. 多余的缓冲区
    5. 15%蔗糖溶液
    6. 50%蔗糖溶液
    7. 3%PFA

设备

  1. PIPETMAN1-20μl(Sartorius,型号:Proline Plus Plus,目录号:728030)
  2. PIPETMAN20-200μl(Sartorius,型号:Proline Plus Plus,目录号:728060)
  3. PIPETMAN100-1,000μl(Sartorius,型号:Proline Plus,目录号:728070)
  4. 水浴(JULABO,型号:TW20)
  5. 二级层流罩(FASTER,型号:SafeFAST Elite)
  6. 细胞培养箱(Heraeus,型号:HERACell)
  7. 离心机(Eppendorf,型号:5415 R)
  8. 倒置显微镜(尼康仪器,型号:Eclipse TS100)
  9. 微孔板光度计(TECAN,型号:Infinite M200)
  10. Dounce匀浆器(DWK Life Sciences,Kimble,目录号:885300-0002)
  11. 陶瓷珠管Lysing矩阵D(MP生物医学,目录号:116913050)
  12. FastPrep-24 MP设备(MP Biomedicals,目录号:116004500)
  13. 超速离心机(Beckman Coulter)
  14. 转子(Beckman Coulter,型号:SW 41 Ti)
  15. MultiScreenHTS真空歧管(Merck,目录号:MSVMHTS00)
  16. 可选:密度梯度分离器(Teledyne Isco)

软件

  1. GraphPad Prism软件

程序

以下程序描述了用于检测嘌呤霉素与核糖体缔合的Ribo-ELISA(David等人,2012)的第一次应用:当时尚不清楚嘌呤霉素结合是否限于翻译核糖体。
吐根碱或CHX都可以用作延长抑制剂来冻结多核糖体。与CHX不同,依米丁不可逆地抑制翻译,并且在整个过程中不需要维护。
第1天:

  1. 预温的生长培养基(见食谱),PBS和37℃的水浴中的胰蛋白酶-EDTA。在70%-80%汇合处将两个175cm 2的细胞的胰蛋白酶消化。取出培养基,用10ml温热的PBS洗涤,加入5ml胰蛋白酶-EDTA并在37℃下保持5分钟。用5毫升生长培养基重悬细胞。
  2. 接种两个175cm2烧瓶,每烧瓶含有1×10 7个HeLa细胞(Corning),每个烧瓶含有15ml温生长培养基。孵育30小时,让HeLa细胞附着和增殖。
  3. 蔗糖梯度(见视频1)。准备15%和50%的蔗糖溶液(见食谱)。在13.2ml聚丙烯离心管(Beckman Coulter)中加入5ml 50%蔗糖溶液。倾斜管(几乎90°),并小心地添加5毫升的15%蔗糖溶液的顶部(慢慢地加入沿管壁流动的溶液,并慢慢地放回管垂直)。用Parafilm密封试管,轻轻将试管水平放置在试验台上。等待1-2小时,然后放回垂直位置。准备好15-50%的蔗糖梯度。存放在冰箱O.N.
    注意:其他方法可用于准备渐变。方便的选择是冻融方法,该方法需要从最高蔗糖浓度(50%,40%,30%,20%,10%w / v蔗糖)开始连续冷冻(-80℃)2ml层状蔗糖溶液解决方案)(Bastide等,2017)。

    视频1

第2天:

  1. 在倒置显微镜下检查HeLa细胞以确保正确汇合(在60-70%之间)。
  2. 用10ml补充有100μg/ ml CHX(或20μg/ ml依米丁)的温热生长培养基直接加入到培养基中孵育细胞5分钟。
  3. 取出培养基,用10毫升温热的PBS洗两次。加入5ml胰蛋白酶-EDTA并将细胞放回培养箱中5分钟,然后用5ml生长培养基重悬细胞,在400×g离心5分钟并用10μl洗涤沉淀两次毫升冰冷的PBS(400xg / 5分钟/ 4℃)(两种溶液都补充有100μg/ ml的CHX,参见食谱)。
    注意:从这一步应该使用RNase免费提示。
  4. 通过离心(400×g / 5分钟/ 4℃)沉淀细胞。重悬细胞1毫升冰冷的匀浆缓冲(见食谱)。
  5. 将细胞悬液转移到冰上的陶瓷珠管(Lysing matrix D)。
    注意:重要的是,当使用陶瓷珠裂解细胞时,NP-40在匀浆缓冲液中不是必需的:以相似的效率回收核糖体。因此,核糖体ELISA可以在没有洗涤剂的情况下进行。
  6. 在室温下涡旋20秒(使用FastPrep-24 MP装置)。
    注意:代替陶瓷珠,细胞可以在冰冷的匀浆缓冲液中使用嵌入在冰中的Dounce匀浆器抚摸10次。
  7. 将上清液转移至1.5ml Eppendorf管中。
    在13,000×g×4℃下旋转10分钟
  8. 仔细收集上清。
    注意:如果上清液不完全清除,请重新离心。
  9. 在冷的15-50%蔗糖梯度的顶部,沿着管壁缓慢地铺上1ml澄清裂解物。用另一个样品或充满水的管子平衡转子。在4℃下以151,263×g(35,000rpm)(Beckman Coulter,转子SW41.Ti)旋转2.5小时。
  10. 分级分离(1ml级分)并使用梯度系统自动化(Teledyne Isco)在254nm处连续测量吸光度。你会得到11分数。
    注意:如果您无法访问自动渐变收集器,则可以以传统的方式收集分数;从顶部手动。使用一个100毫升PIPETMAN 1毫升提示。将活塞推至最大1毫升,并将尖端与弯液面接触。小心地通过缓慢释放吸管活塞来收集1ml蔗糖。始终保持尖端与弯月面接触。把每个部分放在一个干净的1.5毫升试管中(11个部分)。
  11. 向96孔Millipore Multiscreen HTS PVDF板(11孔,泳道1)的孔中加入100μl的各部分。在以下泳道中重复该操作五次以获得两个实验条件(抗嘌呤霉素染色和抗-P蛋白染色)的技术重复(每个部分3个孔)。加载相同的板作为对照('无嘌呤霉素标记')。在冰上孵育板10分钟,然后使用MultiScreenHTS真空歧管对液体进行抽真空。
  12. 加200微升/孔的多聚核糖缓冲液(见食谱),再次抽真空。
  13. 嘌呤霉素标记。向实验板中加入100μl含有多聚核糖体缓冲液的PMY(91μM)和100μl浓度为100μM的多聚核糖体缓冲液(对照板)。在冰上孵育10分钟。同时,跳过这一步为了生成'没有嘌呤霉素标签'控制板。
  14. 轻轻地冲洗水槽,简单地倒在纸巾上。加入200微升多聚核糖体缓冲液,再次弹出平板。重复此操作两次。
  15. 的固定。在引擎盖内加入100μl3%多聚甲醛(PFA)(见食谱)。室温(RT)孵育10分钟。
    正确处理PFA废物 注意:我们在原始协议中使用了这种固定步骤来模仿细胞上的RPM程序。然而,这一步并不需要检测嘌呤化新生链。
  16. 用200μlPBS洗3次。
  17. 阻断膜。用100μl的StartingBlock TM阻断缓冲液孵育30分钟。真空。
    注意:在原始程序中,StartingBlock TM提供了非常低的背景Ab绑定。使用不同的Abs,其他阻塞缓冲区可能会更好。
  18. 1抗体用50μl2.5μg/ ml抗PMy单克隆抗体(在StartingBlock TM中)在室温孵育1小时或抗核糖体抗体(1 / 5,000,在StartingBlock TM )
  19. 用200μlPBS洗3次。
  20. 二抗。与50μl二抗HRP缀合物,用于PMY染色的兔抗小鼠和用于核糖体P染色的山羊抗人(在StartingBlock TM中为1/100)孵育1小时。
  21. 用200μlPBS洗3次。
  22. 信号产生。向每个孔中加入100μlTMB底物(KPL)。当一些蓝色变成可见的时候,用50μl的1.6%HCl停止反应。将每个孔中的液体转移到透明底部的塑料板上。然后,测量在450纳米的吸光度。
  23. 定量通过使用来自对照板的未标记孔的平均值并从测试值中减去它来确定背景染色的水平。在图示的例子中,使用GraphPad Prism软件绘制数据(图1)。


    图1.应用于检测PMY与翻译核糖体结合的核糖体ELISA的示意图(来自David等人,2012)。将用吐根碱孵育15分钟的HeLa细胞或HeLa细胞裂解并在15-50%蔗糖梯度上分级分离。将部分结合到PVDF 96-孔板上,并与PMY一起孵育,这导致核糖体催化的puromycylation。通过A 260级分或通过ELISA检测核糖体蛋白质(通过人类自身免疫Ab检测到的核糖体P蛋白(这里分解成3种已知物种)),其确定60S亚单位和单体(80S)为绑定到PVDF(部分4和5)。使用12-D10通过ELISA对PMY进行纯化,并清楚地表明单体和游离60S亚单位不与PMY稳定缔合,这需要新生链。

数据分析

对于数据分析,我们以前使用GraphPad Prism软件。以下文章介绍了一个例子: David ,2012

笔记

Polysome配置文件可能会有很大的变化,取决于细胞系和翻译率。

食谱

  1. 磷酸盐缓冲盐水(PBS)
    210.0mg / L KH 2 PO 4 4 9,000毫克/升NaCl
    726.0 mg / L Na 2 HPO 4•7H 2 O(Life Technologies)
  2. 生长介质
    Dulbecco改良Eagle谷氨酰胺培养基(赛默飞世尔科技)
    1%青霉素/链霉素
    7.5%胎牛血清(FBS)
  3. 匀浆缓冲液
    50mM Tris-HCl pH7.5
    5mM MgCl 2 2/2 25 mM KCl
    0.2M蔗糖(Sigma-Aldrich)
    0.5%NP-40(赛默飞世尔科技)
    100μg/ ml CHX(Sigma-Aldrich)
    无EDTA蛋白酶抑制剂(罗氏)
    10 U / ml RNase Out(生命技术)
    DEPC水(赛默飞世尔科技)
  4. 多余的缓冲区
    50mM Tris-HCl pH 7.5
    5mM MgCl 2 2/2 25 mM KCl
    0.2M蔗糖(Sigma-Aldrich)
    100μg/ ml CHX(Sigma-Aldrich)
    无EDTA蛋白酶抑制剂(罗氏)
    10 U / ml RNase Out(生命技术)
    DEPC水(赛默飞世尔科技)
  5. 15%蔗糖溶液
    15%蔗糖(m / v)
    50mM Tris-HCl pH 7.5
    5mM MgCl 2 2/2 25 mM KCl
    100μg/ ml CHX(Sigma-Aldrich)
    无EDTA蛋白酶抑制剂(罗氏)
    10 U / ml RNase Out(生命技术)
    DEPC水(赛默飞世尔科技)
  6. 50%蔗糖溶液
    50%蔗糖(m / v)
    50mM Tris-HCl pH 7.5
    5mM MgCl 2 2/2 25 mM KCl
    100μg/ ml CHX(Sigma-Aldrich)
    无EDTA蛋白酶抑制剂(罗氏)
    10 U / ml RNase Out(生命技术)
    DEPC水(赛默飞世尔科技)
  7. 3%PFA
    在PBS 1x
    中稀释原液(16%)

致谢

JWY得到国立过敏和传染病研究所校内研究室的支持。 AD得益于Fondation pour la RechercheMédicale,Ligue contre le Cancer和CancéropôleGSO的慷慨资助。作者声明不存在利益冲突或利益冲突。

参考

  1. Bastide,A.,Peretti,D.,Knight,JR,Grosso,S.,Spriggs,RV,Pichon,X. Sbarrato,T.,Roobol,A.,Roobol,J.,Vito,D.,Bushell,M von der Haar,T.,Smales,CM,Mallucci,GR和Willis,AE(2017)。 RTN3是一种新型的冷诱导蛋白,能够介导RBM3的神经保护作用。 > Curr Biol CB 27:638-650。
  2. David,A.,Dolan,B.P.,Hickman,H.D.,Knowlton,J.J.,Clavarino,G.,Pierre,P.,Bennink,J.R。和Yewdell,J.W。(2012)。 通过核糖体结合的新生链嘌呤化进行可视化的核翻译
  • 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: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
引用:Bastide, A., Yewdell, J. W. and David, A. (2018). Determining Ribosome Translational Status by Ribo-ELISA. Bio-protocol 8(1): e2670. DOI: 10.21769/BioProtoc.2670.
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