细胞中甘油二酯含量的量化分析

Fikadu  G.  Tafesse; Karin  Strijbis; Hidde  L.  Ploegh

doi:10.21769/BioProtoc.1202

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Peer-reviewed

Quantitative Analysis of Cellular Diacylglycerol Content

细胞中甘油二酯含量的量化分析

FT Fikadu G. Tafesse

KS Karin Strijbis

HP Hidde L. Ploegh email

发布: 2014年08月05日第4卷第15期 DOI: 10.21769/BioProtoc.1202 浏览次数: 11947

评审: Hong-guang XiaFanglian He

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参见作者原研究论文

The authors used this protocol in:

Cover of PLOS Pathogens, featuring study using the protocol.

Jun 2013

实验方案合集

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Abstract

Diacylglycerol (DAG) is a bioactive lipid with diverse biological roles. DAG transiently accumulates in a membrane upon receipt of an appropriate stimulus that activates phospholipase C to cleave phospholipids. The resulting hydrolysis product DAG binds to proteins such as protein kinase C to initiate a variety of downstream cellular processes. DAG kinases attenuate such responses by converting DAG to phosphatidic acid.

This protocol describes an assay designed to quantify cellular DAG levels. The assay exploits the enzymatic conversion of DAG (sn-1,2-diacylglycerol) to phosphatidic acid (1,2-diacyl- sn-glycerol-3-phosphate) in conjunction with the incorporation of a radiolabeled phosphate group by DAG kinase (Figure 1). This assay was described in (Strijbis et al., 2013).

Figure 1. The enzymatic conversion of DAG to phosphatidic acid by DAG kinase

Materials and Reagents

Cells (~2 x 10⁶ cells)
Octyl-β-D-glucoside (Sigma-Aldrich, catalog number: O8001 )
Cardiolipin (Sigma-Aldrich, catalog number: C5646 )
Diethylenetriaminepenta acetic acid (DETAPAC) (Sigma-Aldrich, catalog number: D6518 )
Imidazole (Sigma-Aldrich, catalog number: I5513 )
NaCl (Sigma-Aldrich, catalog number: S7653 )
MgCl₂ (Sigma-Aldrich, catalog number: M8266 )
EGTA (Boston Bio Products, catalog number: BM-151 )
DTT (Sigma-Aldrich, catalog number: D0632 )
DAG kinase (Sigma-Aldrich, catalog number: D3065 )
γ³³-ATP (PerkinElmer, catalog number: NEG302H001MC )
DAG (Avanti Polar Lipids)
Phosphatidic acid (Sigma-Aldrich, catalog number: P9511 )
Chloroform (Thermo Fisher Scientific, catalog number: C298-50 )
Methanol (Thermo Fisher Scientific, catalog number: BP1105 )
Acetic acid (Sigma-Aldrich, catalog number: 695092 )
N₂ gas (Middlesex Gases & Technologies)
Acetone (Thermo Fisher Scientific, catalog number: S70090 )

Equipment

Vortex
Centrifuge
Thin layer chromatography (TLC) (Whatman, catalog number: 4860-820 ) equipment (plates, developing tank)
Phospho-scanner (e.g. Fujifilm Corporation, model: BAS-2500 )
Phospho-imaging screens (e.g. Fujifilm Corporation, model: BAS-MS )

Procedure

English

中文翻译

文章信息

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如何引用

Readers should cite both the Bio-protocol article and the original research article where this protocol was used:

Tafesse, F. G., Strijbis, K. and Ploegh, H. L. (2014). Quantitative Analysis of Cellular Diacylglycerol Content. Bio-protocol 4(15): e1202. DOI: 10.21769/BioProtoc.1202.
Strijbis, K., Tafesse, F. G., Fairn, G. D., Witte, M. D., Dougan, S. K., Watson, N., Spooner, E., Esteban, A., Vyas, V. K., Fink, G. R., Grinstein, S. and Ploegh, H. L. (2013). Bruton's Tyrosine Kinase (BTK) and Vav1 contribute to Dectin1-dependent phagocytosis of Candida albicans in macrophages. PLoS Pathog 9(6): e1003446.