Extraction of Chloroplast Proteins from Transiently Transformed Nicotiana benthamiana Leaves
从瞬时转化的本氏烟草中提取叶绿体蛋白质
发布: 2014年09月20日第4卷第18期 DOI: 10.21769/BioProtoc.1238 浏览次数: 20931
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参见作者原研究论文
The authors used this protocol in:
Feb 2014
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Abstract
This rapid protocol allows the extraction of chloroplast enriched proteins from Nicotiana benthamiana (N. benthamiana) leaves that were transiently transformed to express an epitope tagged protein of interest. Thus, it can serve as a tool to study the chloroplastidic localization of the protein of interest when it is combined with western-blot analysis.
Agrobacterium-mediated transformation (Agroinfiltration, Romeis et al., 2001) is used to transiently express a protein carrying an epitope tag in tobacco leaves. Here, co-infiltration with an Agrobacterium strain harboring 19 K from soil-borne wheat mosaic virus suppresses posttranscriptional gene silencing and therefore increases transformation efficiency (Te et al., 2005).
The chloroplast isolation of the transformed leaves is based with modifications on Romeis et al. (2001), and includes mechanical breakage of cell wall and membranes, the removal of unbroken tissue by filtration and the separation of intact chloroplasts by centrifugation through a Percoll layer.
Materials and Reagents
- Nicotiana benthamiana plants
- Agrobacterium tumefaciens (A. tumefaciens) strain GV3101 carrying a recombinant binary plasmid with the gene of interest fused to an epitope tag
- Agrobacterium tumefaciens strain GV3101 harboring K19 (Te et al., 2005)
- Tryptone (AppliChem GmbH, catalog number: 403682 .1210)
- Yeast extract (AppliChem GmbH, catalog number: A1552 , 0500)
- Sucrose (AppliChem GmbH, catalog number: A2211 , 0500)
- MgSO4 (Merck KGaA, catalog number: 105886 )
- Appropriate antibiotic
- Agar (Carl Roth, catalog number: 2266.2 )
- Acetosyringone (Sigma-Aldrich, catalog number: D134406 )
- MES buffer (Carl Roth, catalog number: 4256.3 )
- Liquid nitrogen
- Percoll (GE healthcare, catalog number: 17-0891-01 )
- EDTA (Carl Roth, catalog number: 8040.1 )
- DTT (AppliChem GmbH, catalog number: A2948 )
- MgCl2 (Merck KGaA, catalog number: 105833 )
- Glycerol (AppliChem GmbH, catalog number A3739 ,0500)
- Hepes (Carl Roth, catalog number: 9105 )
- Sorbitol (Merck KGaA, catalog number: 56755 )
- Serum bovine albumin (Carl Roth, catalog number 8076.2 )
- Protease inhibitor cocktail Complete Mini EGTA-free (Roche Diagnostics, catalog number: 11 836 153 001 )
- YEP medium (see Recipes)
- Agromix buffer (see Recipes)
- Isolation buffer (see Recipes)
- Protein extraction buffer (see Recipes)
Equipment
- 1 ml syringe (Terumo Medical Corporation, catalog number: BS-01H )
- Centrifuge cooled at 4 °C
- 47 μm nylon mesh (Carl Roth, catalog number: XA63.1 )
- 2 ml glass homogenizer with round bottom shape (A. Hartenstein, catalog number: HOG2 )
- Light microscope with phase contrast condenser and phase contrast compatible objective lens, such as Objective LD A-Plan 40x/0.5 Ph2 (ZEISS)
- Incubator shaker
- Rotary shaker
- Photospectrometer for OD600
- 1.5 ml reaction tubes (SARSTEDT AG, catalog number 72.706 )
- 50 ml conical centrifuge tubes (SARSTEDT AG, catalog number 62.548.004 )
Procedure
文章信息
版权信息
© 2014 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
- Klinkenberg, J. (2014). Extraction of Chloroplast Proteins from Transiently Transformed Nicotiana benthamiana Leaves. Bio-protocol 4(18): e1238. DOI: 10.21769/BioProtoc.1238.
- Klinkenberg, J., Faist, H., Saupe, S., Lambertz, S., Krischke, M., Stingl, N., Fekete, A., Mueller, M. J., Feussner, I., Hedrich, R. and Deeken, R. (2014). Two fatty acid desaturases, STEAROYL-ACYL CARRIER PROTEIN Delta9-DESATURASE6 and FATTY ACID DESATURASE3, are involved in drought and hypoxia stress signaling in Arabidopsis crown galls. Plant Physiol 164(2): 570-583.
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分类
植物科学 > 植物细胞生物学 > 细胞器分离
植物科学 > 植物生物化学 > 蛋白质 > 分离和纯化
细胞生物学 > 细胞器分离 > 叶绿体
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