In organello Protein Synthesis
细胞器的蛋白质合成
发布: 2014年06月20日第4卷第12期 DOI: 10.21769/BioProtoc.1157 浏览次数: 13222
评审: Ru Zhang
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参见作者原研究论文
The authors used this protocol in:
May 2013
Abstract
In organello protein synthesis method allows the analysis of mitochondrial translation products. The principle of this method relies on incubation of isolated intact mitochondria with radiolabeled amino acids such as 35S methionine. After protein synthesis, the radiolabeled translation products are subsequently separated by SDS polyacrylamide gel electrophoresis and analysed by autoradiography. For in organello analysis of protein synthesis, the isolated intact mitochondria must retain their bioenergetics capacity, and in consequence be fully functional and able to perform coupled respiration. This in turn requires a quick and gentle purification of mitochondria during their isolation.
Keywords: In organello method (在organello方法)
Protein synthesis (蛋白质的合成)
Mitochondria (线粒体)
Arabidopsis thaliana (拟南芥)
Materials and Reagents
- Youngest leaves harvested from 9- to 10-week-old Arabidopsis thaliana
- Common chemicals
Klorin, NaCl, Tween-20, sucrose, tetrasodiumpyrophosphate, PVP-40, EDTA, KH2PO4, sodium ascorbate, L-cysteine, TES, BSA, GTP, mannitol, KCl, DTT, HEPES, MgCl2, sodium acetate, ADP, malic acid, pyruvate, puromycin, L-methionine, isopropanol, acetic acid, Commassie Blue G-250, glycerol, SDS, Tris, β-mercaptoethanol, bromophenol blue - Sand (50-70 mesh particle size) (Sigma-Aldrich, catalog number: 274739 )
- Percoll (pH 8.5-9.5) (25 °C) (Sigma-Aldrich, catalog number: P1644 )
- L-35S methionine (HARTMANN ANALYTIC, catalog number: SRM-01H )
- Amino acid mixture without L-methionine (Promega Corporation, catalog number: L9961 )
- DC Protein Assay Kit (Bio-Rad Laboratories, catalog number: 500-0112 )
- 12% SDS-polyacrylamide gel (Leammli electrophoresis system)
- 10% chlorox solution (see Recipes)
- Grinding medium (see Recipes)
- 2x wash buffer (see Recipes)
- Synthesis mix (see Recipes)
- Stop solution (see Recipes)
- Isopropanol fixing solution (see Recipes)
- Rapid Coomassie Blue G-250 staining solution (see Recipes)
- Destaining solution (see Recipes)
- 1x solubilzation buffer (see Recipes)
Equipment
- Mortar
- Miracloth (Calbiochem®, catalog number: 475855-1R )
- Polycarbonate centrifuge tubes with round bottom (30 ml and 90 ml)
- 1.5 ml microcentrifuge tubes (SARSTEDT AG, catalog number: 72.690.001 )
- Paint brushes
- Tubes with round bottom (SARSTEDT AG, catalog number: 55.484.001 )
- Pasteur pipette
- Gradient former model 485 (Bio-Rad Laboratories, catalog number: 165-4120 )
- Peristaltic pump –PumpP-1 (GE Healthcare, catalog number: 18-111--91 )
- Centrifuges 1K15 and 3K18 (Sigma-Aldrich)
- Microcentrifuges (Eppendorf, catalog number: 5452 000.018 )
- Incubator shaker (IKA KS4000i control shaker, catalog number: 3510001 )
- Spectrophotometer UV-1800 (Schimadzu, catalog number: 206-25400-32 )
- SDS-PAGE system - Mini-Protean Tetra Cell (Bio-Rad Laboratories, catalog number: 165-8000 )
- Slab Gel dryer SGD5040 (Thermo Fisher Scientific, catalog number: SGD5040-230 )
- Radioactive room
- Carestream Kodak BioMax MR film (Sigma-Aldrich, catalog number: Z353949-50EA )
Procedure
文章信息
版权信息
© 2014 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Kwasniak-Owczarek, M. and Janska, H. (2014). In organello Protein Synthesis. Bio-protocol 4(12): e1157. DOI: 10.21769/BioProtoc.1157.
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分类
植物科学 > 植物生物化学 > 蛋白质
细胞生物学 > 细胞器分离 > 线粒体
分子生物学 > 蛋白质 > 检测
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