Protein Degradation Assays in Arabidopsis Protoplasts
利用拟南芥原生质体系统检测蛋白质的降解
发布: 2015年02月20日第5卷第4期 DOI: 10.21769/BioProtoc.1397 浏览次数: 16407
评审: Zhaohui LiuCindy AstAnonymous reviewer(s)
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参见作者原研究论文
The authors used this protocol in:
Jun 2014
Abstract
Plant transformation and exogenous protein expression is essential for molecular biology and biotechnology. Current approaches of stable plant transformation might be problematic and very time-consuming. Because of this, transient expression in protoplasts has become valuable alternative, being less cost and time-effective at the same time. Excellent for eukaryotic proteins, representing a natural cell habitat, protoplast isolation is widely used in protein interaction visualization techniques, like BiFC (Bimolecular fluorescence complementation) and FRET (Förster resonance energy transfer). In this protocol we present a another use of Arabidopsis protoplast in protein degradation assay, proving its high versatility as a tool in proteomics.
Transient expression (瞬时表达)
Protoplasts (原生质体)
ABA signalling (ABA信号)
Ethylene biosynthesis (乙烯的生物合成)
Materials and Reagents
- 3-week old Arabidopsis plants
- Mannitol (BDH Prolabo, catalog number: 25311 )
- CaCl2 (POCH, catalog number: M00015143 )
- KCl (USB, catalog number: 20598 )
- 2-(N-morpholino)ethanesulfonic acid (MES) (LabEmpire, catalog number: MES503 )
- NaCl (POCH, catalog number: BA4121116 )
- Polyethylene glycol (PEG) 4000 (Sigma-Aldrich, catalog number: 81240 )
- Cellulase (SERVA Electrophoresis GmbH, catalog number: 16419 )
- Macerozyme (SERVA Electrophoresis GmbH, catalog number: 28302 )
- GenEluteTM HP Plasmid Midiprep Kit (Sigma-Aldrich, catalog number: PLD35 )
- Benzyloxycarbonyl-L-leucyl-L-leucyl-L-leucinal, Z-Leu-Leu-Leu-al (MG132) (Sigma-Aldrich, catalog number: M7449 )
- Dimethyl sulfoxide (DMSO) (Sigma-Aldrich, catalog number: D8418 )
- HEPES buffer (Sigma-Aldrich, catalog number: H3375 )
- MgCl2 (USB, catalog number: 18641 )
- Dithiothreitol (DTT) (Sigma-Aldrich, catalog number: D0632 )
- Phenylmethylsulfonyl fluoride (PMSF) (Sigma-Aldrich, catalog number: P7626 )
- Protease inhibitors (Roche Diagnostics, catalog number: 11873580001 )
- Enzyme solution (see Recipes)
- W5 (see Recipes)
- W1 (see Recipes)
- MMg (see Recipes)
- 40% PEG (see Recipes)
- Protein isolation buffer (see Recipes)
Equipment
- Light microscope (Nikon Corporation)
- Hemocythometer (Sigma-Aldrich)
- Tape (Scotch® MagicTM)
- Stainless steel forceps (Sigma-Aldrich)
- Scissors
- 10 ml pipette
- 50 ml tubes
- 90 mm Petri dishes
- Amicon Ultra-15 centrifugal filter unit (Millipore)
- NanoDrop spectrophotometer
- Tabletop centrifuge
- Horizontal shaker
Procedure
文章信息
版权信息
© 2015 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Mituła, F., Kasprowicz-Maluśki, A., Michalak, M., Marczak, M., Kuczyński, K. and Ludwików, A. (2015). Protein Degradation Assays in Arabidopsis Protoplasts. Bio-protocol 5(4): e1397. DOI: 10.21769/BioProtoc.1397.
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分类
植物科学 > 植物细胞生物学 > 细胞分离
植物科学 > 植物分子生物学 > 蛋白质
细胞生物学 > 组织分析 > 组织分离
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