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
发布: 2012年11月05日第2卷第21期 DOI: 10.21769/BioProtoc.282 浏览次数: 27996
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参见作者原研究论文
The authors used this protocol in:
Mar 2012
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
- Methionine-free medium DMEM (Sigma-Aldrich, catalog number: D0422 )
- Fetal calf serum (Hyclone, catalog number: SV30160.03 )
- Fetal bovine serum (FBS)
- Penicillin/streptomycin/glutamine
- Phosphate buffered saline (PBS) (Life Technologies, Invitrogen™, catalog number: 10010-056 )
- Protein assay kit (DC Protein Assay Kit I-500) (Bio-Rad, catalog number: 0111EDU )
- Protein A/G PLUS-Agarose (Santa Cruz Biotechnology, catalog number: sc-2003 )
- EasyTaq -[35S]-Methionine, 5 mCi (185 MBq), stabilized aqueous solution (Perkinelmer, catalog number: NEG709A005MC )
- Hybond ECL Nitrocellulose Membrane (Amersham, catalog number: RPN68D )
- Kodak Biomax XAR film (Sigma-Aldrich, catalog number: F5763 )
- Immobilon Western Chemiluminescent HRP Substrate (EMD Millipore, catalog number: WBKLS0500 )
- Anti-MDM2 (Santa Cruz)
- HEPES
- NaCl
- Glycerol
- Triton X-100
- MgCl2
- EGTA
- Na4P2O7
- NaF
- Aprotinin
- Leupeptin
- PMSF
- Na3VO4
- 2-mercaptoethanol
- Acrylamide
- Bromophenol blue
- Ammonium persulfate (APS)
- Lysis buffer (see Recipes)
- HNTG buffer (see Recipes)
- Protein A or G agarose beads (see Recipes)
- 2x laemmli buffer (see Recipes)
- SDS-polyacrylamide gel (see Recipes)
- 10x electrophoresis buffer (see Recipes)
- 1x transfer buffer (see Recipes)
Equipment
- Centrifuges
- Vortexer
- Tissue culture hood and incubator
- Radioactive material and room
- Western-Blot apparatus
- Developer
- Hamilton syringe
- Spectrophotometer
- Rocker
- T25 flask
Procedure
文章信息
版权信息
© 2012 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
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.
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分类
生物化学 > 蛋白质 > 标记
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