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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, 11588 views, 1 Q&A
DOI: https://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)

[Abstract] 该实验方案的中文版正在准备中...

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.

材料和试剂

  1. 不含甲硫氨酸的培养基DMEM(Sigma-Aldrich,目录号:D0422)
  2. 胎牛血清(Hyclone,目录号:SV30160.03)
  3. 胎牛血清(FBS)
  4. 青霉素/链霉素/谷氨酰胺
  5. 磷酸盐缓冲盐水(PBS)(Life Technologies,Invitrogen TM,目录号:10010-056)
  6. 蛋白测定试剂盒(DC蛋白测定试剂盒I-500)(Bio-Rad,目录号:0111EDU)
  7. 蛋白A/G PLUS-琼脂糖(Santa Cruz Biotechnology,目录号:sc-2003)
  8. EasyTaq - [35 S] - 甲硫氨酸,5mCi(185MBq),稳定的水溶液(Perkinelmer,目录号:NEG709A005MC)
  9. Hybond ECL硝化纤维素膜(Amersham,目录号:RPN68D)
  10. Kodak Biomax XAR膜(Sigma-Aldrich,目录号:F5763)
  11. Immobilon Western化学发光HRP底物(EMD Millipore,目录号:WBKLS0500)
  12. 抗MDM2(圣克鲁斯)
  13. HEPES
  14. NaCl
  15. 甘油
  16. Triton X-100
  17. MgCl 2
  18. EGTA
  19. Na <4>

  20. NaF
  21. 抑肽酶
  22. 亮肽素
  23. PMSF
  24. Na 3 VO 4
  25. 2-巯基乙醇
  26. 丙烯酰胺
  27. 溴酚蓝
  28. 过硫酸铵(APS)
  29. 裂解缓冲液(见配方)
  30. HNTG缓冲区(见Recipes) 
  31. 蛋白A或G琼脂糖珠(见配方)
  32. 2x laemmli缓冲液(参见配方)
  33. SDS-聚丙烯酰胺凝胶(参见配方)
  34. 10x电泳缓冲液(参见配方)
  35. 1x传输缓冲区(请参阅配方)

设备

  1. 离心机
  2. Vortexer
  3. 组织培养罩和孵化器
  4. 放射性物质和房间
  5. 西方印迹设备
  6. 开发人员
  7. Hamilton注射器
  8. 分光光度计
  9. 摇杆
  10. T25烧瓶

程序

  1. 代谢标记
    1. 在补充有10%胎牛血清和青霉素/链霉素/谷氨酰胺的30ml不含甲硫氨酸的培养基(DMEM)中洗涤1×10 7个细胞/样品3次。
    2. 在T25烧瓶中的10ml无甲硫氨酸培养基中收获细胞60分钟
    3. 将细胞重悬在含有250uCi /样品[35S] - 甲硫氨酸的10ml培养基中30分钟。
    4. 在10ml PBS中洗涤细胞两次,并在1,200rpm离心10分钟
    5. 通过在4℃下每隔5分钟涡旋15秒,用500μl裂解缓冲液裂解细胞30分钟。
    6. 在10,000×g离心30秒以在4℃沉淀DNA
    7. 通过DC蛋白测定试剂盒I确定上清液中的蛋白质水平
    8. 蛋白质定量后,取等量的蛋白质提取物(约1mg)进行免疫沉淀

  2. 免疫沉淀
    1. 通过在eppendorf管中用3μg针对目的蛋白的抗体(此处为30μl抗MDM2抗体)旋转,在4℃下孵育等量的裂解物。
    2. 旋转裂解物(以便在旋转后收集盖中的所有液滴)。
    3. 通过吸取尖端(边缘已经切断)(见配方3)加入30微升蛋白质A/G PLUS-琼脂糖浆体积,并在摇床上在4℃下孵育2小时。
    4. 用500μlHNTG缓冲液洗涤免疫沉淀(珠子)4次,通过在4℃下以10,000xg离心珠子30秒。
    5. 最后用Hamilton注射器吸出HNTG缓冲液
    6. 在珠上加入30μlLaemmli缓冲液2x
    7. 将样品煮沸5分钟。

  3. 在SDS-聚丙烯酰胺凝胶电泳和放射自显影上分辨目标蛋白
    1. 在变性条件下在SDS-聚丙烯酰胺凝胶电泳上分离蛋白质
    2. 将其转移到硝酸纤维素膜上,并且在暴露于X-AR膜之后,将新合成的[35 S]甲硫氨酸蛋白质可视化。
    3. 通过与适当的一抗和二抗温育过夜来验证免疫沉淀负载(参见Western印迹方案)
    4. 用化学发光HRP底物检测试剂盒检测蛋白质

食谱

  1. 裂解缓冲液
    50mM HEPES(pH 7.0)
    150mM NaCl 10%甘油 1%Triton X-100 1.5mM MgCl 2·h/v 1 mM EGTA
    10mM Na 4 PO 4 sub 2 O 7
    +添加临时
    10mM NaF 1 mM DTT
    10μgL 抑肽酶
    10微克L -1 亮肽素
    1mM PMSF
    1mM Na 3 VO 4 sub。
  2. HNTG缓冲区
    50mM HEPES(pH 7.0)
    10%甘油 0.3%Triton X-100 150mM NaCl 1 mM NaVO 4
  3. 蛋白A或G琼脂糖珠
    用PBS洗珠子两次
    用PBS恢复至50%浆料
    (建议将尖端的边缘切成移液管)
  4. 2x laemmli缓冲区
    4%SDS
    20%甘油 10%2-巯基乙醇 0.004%溴酚蓝
    0.125M Tris-HCl
  5. SDS-聚丙烯酰胺凝胶 10%PAGE
    H 2 O 4 ml
    30%丙烯酰胺3.3ml
    1.5M Tris(pH8.8)2.5ml
    10%SDS 0.1ml
    10%过硫酸铵(APS)0.1ml
    10%TEMED 0.012ml
    堆叠
    H O 5.6 ml
    丙烯酰胺(30%)1.7ml
    0.5M Tris(pH8.8)2.5ml
    10%SDS 0.1ml
    10%过硫酸铵(APS)0.125ml
    10%TEMED 0.015ml
  6. 10x电泳缓冲液
    大豆144 g
    Tris碱30 g
    20%SDS 50毫升
    H sub 2 O qsp 1 L
  7. 1x传输缓冲区
    甘氨酸14 g
    Tris碱3 g
    20%乙醇200ml
    H sub 2 O qsp 1 L

致谢

该协议先前已在Nakatake等人(2012)中发表。 这项工作得到了来自"协会资助癌症(Projet libre 2012),Agence Nationale de la Recherche,计划Jeunes Chercheuses和Jeunes Chercheurs,卓越实验室Globule Rouge-Excellence的资金资助的方案"Investissements d' avenir"。HS得到了La Ligue Nationale Contrele Cancer的奖学金的支持。

参考文献

  1. Nakatake,M.,Monte-Mor,B.,Debili,N.,Casadevall,N.,Ribrag,V.,Solary,E.,Vainchenker,W.and Plo, JAK2(V617F)通过在骨髓增生性肿瘤中通过La表达增强MDM2来负调节p53稳定。 Oncogene 31(10):1323-1333。
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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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