Detection of Mitogen-activated Protein Kinase (MAPK) Activation upon Exogenous Chemical Application in Arabidopsis Protoplasts
外源化学物质处理后拟南芥原生质体中促分裂原活化蛋白激酶（MAPK）的检测
发布: 2015年09月20日第5卷第18期 DOI: 10.21769/BioProtoc.1588 浏览次数: 12652
评审: Zhaohui LiuArsalan DaudiAnonymous reviewer(s)
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参见作者原研究论文
The authors used this protocol in:
Nov 2014
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Abstract
The mitogen activated protein kinase cascade is a highly conserved signal pathway in plants. The exogenous chemicals, like hormones, can trigger a series of signalling cascades, including MAPK pathway, to modulate the plant physiology. Upon activation, some MAPKs are phosphorylated. It is important to develop methods that can detect changes in the phosphorylation status of MAPKs in plants when they come in contact with external chemicals. This method describes the exogenous treatment of Arabidopsis protoplasts with Kinetin and subsequent detection of the activated MAPKs. This method is useful for studying the effect of exogenously applied chemical compounds on the MAPK signaling cascade in Arabidopsis.
Keywords: MAP kinase (MAP激酶)
Phosphorylation (磷酸化)
In vitro kinase assay (体外激酶测定)
Signal transduction (信号转导)
Immunoprecipitation (免疫共沉淀)
Materials and Reagents
- Plants: Arabidopsis thaliana (Col-0) obtained from NASC The European Arabidopsis Stock Centre
- Sodium chloride (NaCl) (Carl Roth, catalog number: 3957 )
- MgCl2 (Thermo Fisher Scientific, catalog number: M35 )
- CaCl2 (Thermo Fisher Scientific, catalog number: C69 )
- Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P9541 )
- D-Mannitol (Sigma-Aldrich, catalog number: M4125 )
- MES hydrate (Sigma-Aldrich, catalog number: 8250 )
- Albumin bovine modified Cohn Fraction V, pH 7.0 (BSA) (SERVA Electrophoresis GmbH, catalog number: 11943 )
- Macerozyme ONOZUKA R10 (Yakult Pharmaceutical Industry Co)
- Cellulase ONOZUKA R10 (Yakult Pharmaceutical Industry Co)
- Kinetin (Sigma-Aldrich, catalog number: K3378 )
- Phospho-p44/42 MAPK (ERK1/2) Antibody (pTEpY Antibody) (Cell Signaling Technology, catalog number: 9101 )
- PierceTM ECL Western Blotting Substrate (Thermo Fisher Scientific, catalog number: 32109 )
- Protein free blocking solution (PFBS) (Thermo Fisher Scientific)
- 0.45 µm acetate filter (Carl Roth, catalog number: AH53.1 )
- Petri Dishes (Greiner Bio-One GmbH, catalog number: 628103 )
- Nitrocellulose Hybond ECL membrane (GE Healthcare, Amersham biosciences®, model: RPN82D )
- Stock solutions (see Recipes)
- Enzyme solution (see Recipes)
- W5 solution (see Recipes)
- WI solution (see Recipes)
- 0.5 mM mannitol (see Recipes)
- Kinetin (see Recipes)
- 1 L transfer buffer (see Recipes)
- 1 LTBST (Tris-Buffered Saline and Tween 20) (see Recipes)
- 5x SDS-Loading buffer (see Recipes)
- 10% SDS-PAGE gel (see Recipes)
Equipment
- Phytochambers or light room or greenhouse (23 °C, 16 h dark / 8 h light)
- Sharp razor blades (Carl Roth, catalog number: CK07.1 )
- Polycarbonate dessicator (Carl Roth, catalog number: PK30.1 )
- Nylon Net Filter, Hydrophilic (100 µm, 25 mm) (EMD Millipore, catalog number: NY1H02500 )
- Round bottom 12 ml cell culture polystyrene tubes (Greiner Bio-One GmbH, catalog number: 163160 )
- Swing bucket refrigerated Centrifuge (Eppendorf, model: 6810R )
- Haemocytometer
- SDS-PAGE gel running apparatus (Bio-Rad Laboratories)
- Semi-dry blotting apparatus (Bio-Rad Laboratories)
Procedure
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文章信息
版权信息
© 2015 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Sheikh, A. H. and Sinha, A. K. (2015). Detection of Mitogen-activated Protein Kinase (MAPK) Activation upon Exogenous Chemical Application in Arabidopsis Protoplasts. Bio-protocol 5(18): e1588. DOI: 10.21769/BioProtoc.1588.
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分类
植物科学 > 植物免疫 > 信号感知与传递
生物化学 > 蛋白质 > 活性
细胞生物学 > 基于细胞的分析方法 > 基因表达
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