Quantification of Low Molecular Weight Thiols in Arabidopsis
拟南芥中低分子量硫醇的定量测定
发布: 2016年01月05日第6卷第1期 DOI: 10.21769/BioProtoc.1704 浏览次数: 7432
评审: Samik BhattacharyaMoritz BomerAnonymous reviewer(s)
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参见作者原研究论文
The authors used this protocol in:
Feb 2015
Abstract
Low-molecular-weight (LMW) thiols are a class of highly reactive compounds due to their thiol moiety. They play important roles in the maintenance of cellular redox homeostasis, detoxification, and development. Monobromobimane (mBBr) is weakly fluorescent but selectively reacts with thiols to yield highly fluorescent thioethers (mBSR) products, which is especially useful for the quantification of LMW thiols. The stable mBSR products can be separated by high-performance liquid chromatography (HPLC) equipped with a fluorescent detector. The main cellular LMW thiols are L-cysteine, gamma-glutamylcysteine, and glutathione (GSH). The following protocol describes the extraction and quantification of L-cysteine, gamma-glutamylcysteine, and glutathione from Arabidopsis tissues as reported (Xiang and Oliver, 1998; Zhao et al., 2014; Wang et al., 2015) with minor revision. Modifications may be required if the HPLC system or the C18 column is different.
Keywords: Cysteine (半胱氨酸)
Glutathione (谷胱甘肽)
Arabidopsis (拟南芥)
HPLC (高效液相色谱法)
Fluorescent (荧光)
Materials and Reagents
- 1.5 ml microcentrifuge tube
- 0.2 μm nylon filter (Sigma-Aldrich, catalog number: Z259969 )
- 100 ml glass syringe
- Pipette tip
- Fresh Arabidopsis thaliana tissues (any plant part you want to test, 50-100 mg is sufficient)
- L-cysteine (Sigma-Aldrich, catalog number: 778672 )
- Gamma-glutamylcysteine (γ-Glu-Cys) (Sigma-Aldrich, catalog number: G0903 )
- L-Glutathione reduced (Sigma-Aldrich, catalog number: G4251 )
- Double distilled water (supplied by School of Life Sciences, University of Science and Technology of China)
- Double distilled water
- Hydrochloric Acid (HCl) (Sigma-Aldrich, catalog number: 435570 ) (see Recipes)
- 2-(N-Morpholino)ethanesulfonic acid hydrate (MES) (Sigma-Aldrich catalog number: M8250 ) (see Recipes)
- Ethylenediaminetetraacetic acid disodium salt dihydrate (EDTA.2Na.2H2O) (Sangon Biotech, catalog number: A100105 ) (see Recipes)
- Monobromobimane (mBBr) (Sigma-Aldrich, catalog number: 69898 ) (see Recipes)
- Trifluoroacetic acid (TFA) (Sigma-Aldrich, catalog number: 302031 ) (see Recipes)
- Acetonitrile (OCEANPAK, catalog number: Ac00030281 ) (see Recipes)
Equipment
- Analytic balance (Mettler-Toledo International Inc., model: ML104 )
- Mortar and Pestle
- Refrigerated microcentrifuge (Thermo Fisher Scientific, Eppendorf, model: 5424R )
- 37 °C degree incubator (Shanghai Jinghong Laboratory Instrument, model: GNP-9080 )
- Reverse-phase C18 column (5 µm, 110A, 150 x 4.6 mm) (Phenomenex, model: Gemini C18 column ) or equivalent, guard column (Phenomenex, SecurityGuard Standard, model: AJ0-7597 )
- HPLC equipment (Agilent Technologies, model: 1200 series )
- 200 μl pipette
- pH meter (Thermo Fisher Scientific, Mettler Toledo, model: FE20–FiveEasy PlusTM )
- Ultrasonic cleaner (Shanghai Sonxi Ultrasonic Instrument, model: DS-2510DT )
Software
- Suitable data collection and processing software (Agilent Technologies, model: 1200 ChemStation)
- Standard curve plotting (Microsoft Excel)
Procedure
文章信息
版权信息
© 2016 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Miao, Z., Wang, Z. and Xiang, C. B. (2016). Quantification of Low Molecular Weight Thiols in Arabidopsis. Bio-protocol 6(1): e1704. DOI: 10.21769/BioProtoc.1704.
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分类
植物科学 > 植物生物化学 > 其它化合物
生物化学 > 其它化合物 > 硫醇
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