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To follow protein synthesis, cells should be incubated with radioactive amino acid such as [35S] methionine during mRNA translation. Then, the neosynthetized protein will be identified by an autoradiography after immunoprecipitation with a specific antibody and separation on a polyacrylamide denaturing gel.

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Protein Translation Study – Label Protein with S35 Methionine in Cells
蛋白质转译研究-—用S35蛋氨酸标记细胞中的蛋白质

生物化学 > 蛋白质 > 标记
作者: Salma Hasan
Salma HasanAffiliation: INSERM U1009, Gustave Roussy, Villejuif, France
Bio-protocol author page: a140
 and Isabelle Plo
Isabelle PloAffiliation: INSERM U1009, Gustave Roussy, Villejuif, France
For correspondence: isabelle.plo@gustaveroussy.fr
Bio-protocol author page: a141
Vol 2, Iss 21, 11/5/2012, 10101 views, 1 Q&A, How to cite
DOI: http://dx.doi.org/10.21769/BioProtoc.282

[Abstract] To follow protein synthesis, cells should be incubated with radioactive amino acid such as [35S] methionine during mRNA translation. Then, the neosynthetized protein will be identified by an autoradiography after immunoprecipitation with a specific antibody and separation on a polyacrylamide denaturing gel.

Keywords: Protein synthesis(蛋白质的合成), Translation(翻译), S35 methionine(S35 methionine)

Materials and Reagents

  1. Methionine-free medium DMEM (Sigma-Aldrich, catalog number: D0422)
  2. Fetal calf serum (Hyclone, catalog number: SV30160.03)
  3. Fetal bovine serum (FBS)
  4. Penicillin/streptomycin/glutamine
  5. Phosphate buffered saline (PBS) (Life Technologies, Invitrogen™, catalog number: 10010-056)
  6. Protein assay kit (DC Protein Assay Kit I-500) (Bio-Rad, catalog number: 0111EDU)
  7. Protein A/G PLUS-Agarose (Santa Cruz Biotechnology, catalog number: sc-2003)
  8. EasyTaq -[35S]-Methionine, 5 mCi (185 MBq), stabilized aqueous solution (Perkinelmer, catalog number: NEG709A005MC)
  9. Hybond ECL Nitrocellulose Membrane (Amersham, catalog number: RPN68D)
  10. Kodak Biomax XAR film (Sigma-Aldrich, catalog number: F5763)
  11. Immobilon Western Chemiluminescent HRP Substrate (EMD Millipore, catalog number: WBKLS0500)
  12. Anti-MDM2 (Santa Cruz)
  13. HEPES
  14. NaCl
  15. Glycerol
  16. Triton X-100
  17. MgCl2
  18. EGTA
  19. Na4P2O7
  20. NaF
  21. Aprotinin
  22. Leupeptin
  23. PMSF
  24. Na3VO4
  25. 2-mercaptoethanol
  26. Acrylamide
  27. Bromophenol blue
  28. Ammonium persulfate (APS)
  29. Lysis buffer (see Recipes)
  30. HNTG buffer (see Recipes) 
  31. Protein A or G agarose beads (see Recipes)
  32. 2x laemmli buffer (see Recipes)
  33. SDS-polyacrylamide gel (see Recipes)
  34. 10x electrophoresis buffer (see Recipes)
  35. 1x transfer buffer (see Recipes)

Equipment

  1. Centrifuges
  2. Vortexer
  3. Tissue culture hood and incubator
  4. Radioactive material and room
  5. Western-Blot apparatus
  6. Developer
  7. Hamilton syringe
  8. Spectrophotometer
  9. Rocker
  10. T25 flask

Procedure

  1. Metabolic labeling
    1. Wash 1 x 107 cells/sample in 30 ml methionine-free medium (DMEM) supplemented with 10% fetal bovine serum and penicillin/streptomycin/glutamine for 3 times.
    2. Harvest cells for 60 min in 10 ml methionine-free medium in T25 flask.
    3. Resuspend cells in 10 ml medium containing 250 uCi/sample [35S]-methionine for 30 min.
    4. Wash cells twice in 10 ml PBS and centrifuge them at 1,200 rpm for 10 min.
    5. Lyse the cells with 500 μl of lysis buffer for 30 min by vortexing 15 sec every 5 min at 4 °C.
    6. Centrifuge at 10,000 x g for 30 sec to pellet the DNA at 4 °C.
    7. Determine the protein levels in the supernatant by DC Protein assay kit I.
    8. Take same amount of protein extracts (about 1 mg) for immunoprecipitation after protein quantification.

  2. Immunoprecipitation
    1. Incubate equal amount of lysates onvernight at 4 °C by rotation with 3 μg antibody against protein of interest (here 30 μl of anti-MDM2 antibody) in eppendorf tubes.
    2. Spin down the lysates (in order to collect all the drops in the cap after rotation).
    3. Add 30 μl of Protein A/G PLUS-Agarose slurry volume by pipetting with tips (edge already cut off) (see Recipes 3) and incubate 2 h on a rocker at 4 °C.
    4. Wash immunoprecipitates (beads) with 500 μl HNTG buffer for 4 times by centrifuging beads at 10, 000 x g for 30 sec at 4 °C.
    5. At the end aspirate HNTG buffer with Hamilton syringe.
    6. Add 30 μl of Laemmli buffer 2x on beads.
    7. Boil the sample for 5 min.

  3. Resolving protein of interest on SDS-polyacrylamide gel electrophoresis and autoradiography
    1. Resolve the protein on SDS-polyacrylamide gel electrophoresis under denaturing conditions.
    2. Transfer it onto nitrocellulose membrane and newly synthesized [35S]methionine-protein will be visualized after exposure to X-AR films.
    3. Verify the immunoprecipitation loading by incubating overnight with appropriate primary and secondary antibodies (see Western-blot protocol).
    4. Detect the protein with a chemoluminecent HRP substrate detection kit.

Recipes

  1. Lysis buffer
    50 mM HEPES (pH 7.0)
    150 mM NaCl
    10% glycerol
    1% Triton X-100
    1.5 mM MgCl2
    1 mM EGTA
    10 mM Na4P2O7
    +add extemporary
    10 mM NaF
    1 mM DTT
    10 μg L-1 aprotinin
    10 μg L-1 leupeptin
    1 mM PMSF
    1 mM Na3VO4
  2. HNTG buffer
    50 mM HEPES (pH 7.0)
    10% glycerol
    0.3% Triton X-100
    150mM NaCl
    1 mM NaVO4
  3. Protein A or G agarose beads
    Wash the beads twice with PBS
    Restore to 50% slurry with PBS
    (It is recommended to cut the edge of the tip to pipet)
  4. 2x laemmli buffer
    4% SDS
    20% glycerol
    10% 2-mercaptoethanol
    0.004% bromophenol blue
    0.125 M Tris-HCl
  5. SDS-polyacrylamide gel
    10% PAGE
    H2O 4 ml
    30% Acrylamide 3.3 ml
    1.5 M Tris (pH 8.8) 2.5 ml
    10% SDS 0.1 ml
    10% Ammonium persulfate (APS) 0.1 ml
    10% TEMED 0.012 ml
    STACKING
    H2O 5.6 ml
    Acrylamide (30%) 1.7 ml
    0.5M Tris (pH 8.8) 2.5 ml
    10% SDS 0.1 ml
    10% Ammonium persulfate (APS) 0.125 ml
    10% TEMED 0. 015 ml
  6. 10x electrophoresis buffer
    Glycine 144 g
    Tris base 30 g
    20% SDS 50 ml
    H2O qsp 1 L
  7. 1x transfer buffer
    Glycine 14 g
    Tris base 3 g
    20% Ethanol 200 ml
    H2O qsp 1 L

Acknowledgments

The protocol was previously published in Nakatake et al. (2012). This work was supported by grants from ” Association pour la Recherche sur le Cancer (projet libre 2012), Agence Nationale de la Recherche, programme Jeunes Chercheuses et Jeunes Chercheurs, Laboratory of Excellence Globule Rouge-Excellence is funded by the program “Investissements d’avenir.” HS was supported by fellowships from la Ligue Nationale Contre le Cancer.

References

  1. Nakatake, M., Monte-Mor, B., Debili, N., Casadevall, N., Ribrag, V., Solary, E., Vainchenker, W. and Plo, I. (2012). JAK2(V617F) negatively regulates p53 stabilization by enhancing MDM2 via La expression in myeloproliferative neoplasms. Oncogene 31(10): 1323-1333.


How to cite this protocol: Hasan, S. and Plo-Azevedo, I. (2012). Protein Translation Study – Label Protein with S35 Methionine in Cells . Bio-protocol 2(21): e282. DOI: 10.21769/BioProtoc.282; Full Text



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11/19/2013 10:52:47 PM  

Simply add in culture medium?

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