Improve Research Reproducibility A Bio-protocol resource
Peer-reviewed

Determining Ribosome Translational Status by Ribo-ELISA

采用Ribo-ELISA检测核糖体的翻译状态

AB Amandine Bastide
JY Jonathan W. Yewdell
AD Alexandre David

发布: 2018年01月05日第8卷第1期 DOI: 10.21769/BioProtoc.2670 浏览次数: 7294

评审: Alessandro DidonnaSteven BoeynaemsAnonymous reviewer(s)

download pdf PDF PDF
ask Q&A
引用
收藏
Cited by

参见作者原研究论文

The authors used this protocol in:

Cover of The Journal of Cell Biology, featuring study using the protocol.

Apr 2012

Presubmission Inquiry

实验方案合集

专注于特定技术或应用的、详细的、经过同行评审的实验方案合集

Cancer Research
Immunology
Developmental Biology
Microbiology
Neuroscience
Cell Imaging - A Special Collection for Cell Bio 2023
查看更多 more

相关实验方案

在PC-3细胞中处理5天后的线粒体生成分析

在PC-3细胞中处理5天后的线粒体生成分析

Yili Xu and Joy Y. Feng

2015年01月20日 11651 阅读

生物材料中4-羟基壬烯醛与蛋白质加合物的高灵敏、可重复检测法

生物材料中4-羟基壬烯醛与蛋白质加合物的高灵敏、可重复检测法

T. Blake Monroe and Ethan J. Anderson

2019年10月05日 3392 阅读

用于检测和定量人血浆或血清中抗病毒抗体的优化间接 ELISA 方案：以 SARS-CoV-2 为例的案例研究

用于检测和定量人血浆或血清中抗病毒抗体的优化间接 ELISA 方案：以 SARS-CoV-2 为例的案例研究

Claire Baine [...] Jennifer Serwanga

2023年12月20日 2693 阅读

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) search ELISA (ELISA) search Ribopuromycylation (Ribopuromycylation) search

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

    English
    中文翻译

    文章信息

    版权信息

    © 2018 The Authors; exclusive licensee Bio-protocol LLC.

    如何引用

    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.

    ris ris Download Citation in RIS Format

    分类

    生物化学 > 蛋白质 > 免疫检测

    您对这篇实验方法有问题吗？

    在此处发布您的问题，我们将邀请本文作者来回答。同时，我们会将您的问题发布到Bio-protocol Exchange，以便寻求社区成员的帮助。

    0/150

    tip 提问指南

    + 问题描述

    写下详细的问题描述，包括所有有助于他人回答您问题的信息（例如实验过程、条件和相关图像等）。

    post 发布问题
    0 Q&A
    pen 提交稿件