Determining Ribosome Translational Status by Ribo-ELISA

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 (MµltiGuard^TM 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, Gibco^TM, catalog number: 25300054 )
      
   2. StartingBlock^TM blocking buffer (Thermo Fisher Scientific, Thermo Scientific^TM, 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, Gibco^TM, catalog number: 41966029 )
      
   8. Penicillin/streptomycin (Thermo Fisher Scientific, Gibco^TM, catalog number: 10378016 )
      
   9. Fetal bovine serum (FBS) (Eurobio, catalog number: CVFSVF0101 )
      
   10. Tris-HCl pH 7.5 (Thermo Fisher Scientific, Invitrogen^TM, 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 Scientific^TM, 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, Invitrogen^TM, catalog number: 10777019 )
       
   17. DEPC treated water (Thermo Fisher Scientific, Invitrogen^TM, catalog number: 750023 )
       
   18. 16% paraformaldehyde (Electron Microscopy Sciences, catalog number: 15710 )
       
   19. Glutamine (Thermo Fisher Scientific, Gibco^TM, catalog number: 25030081 )
       
   20. 70% ethanol (Thermo Fisher Scientific, Thermo Scientific^TM, 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