[14C] Linoleic Acid Uptake and Fractionation Assay in Vibrio cholerae
霍乱弧菌中[14C] 亚油酸的摄取和分级分离试验
发布: 2015年12月20日第5卷第24期 DOI: 10.21769/BioProtoc.1682 浏览次数: 7036
评审: Valentine V TrotterElizabeth LibbyAnonymous reviewer(s)
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参见作者原研究论文
The authors used this protocol in:
May 2015
Abstract
The gram-negative curved bacillus Vibrio cholerae (V. cholerae) causes the severe diarrheal illness cholera. The work presented here is to assess whether unsaturated fatty acids (UFAs), such as linoleic acid, have the potential to directly affect proteins involved in DNA binding because they are able to enter the cell. In this protocol, we show how to measure linoleic acid entering V. cholerae when added exogenously and determine whether it is able to enter the cytoplasm. This protocol will quantify how much linoleic acid is able to enter the cell and then identify the amount of linoleic acid that stays in the membrane or ultimately enters the cytoplasm.
Keywords: Vibrio cholerae (霍乱弧菌)
Cholera (霍乱)
Linoleic acid (亚油酸)
Unsaturated fatty acids (不饱和脂肪酸)
Background
Materials and Reagents
- Scintillation vials (Thermo Fisher Scientific, catalog number: 03-337-20 )
- Autoclaved 1.7 ml microcentrifuge tubes (BioExpress, catalog number: C-3262-1 )
- Test Tubes (Thermo Fisher Scientific, catalog number: 14-955E )
- 15 ml conical screw cap tubes (BioExpress, catalog number: C-3394-2 )
- Vibrio cholerae classical biotype strain O395
- 14C linoleic acid (PerkinElmer, catalog number: NEC501050UC )
- Scintillation cocktail (Thermo Fisher Scientific, catalog number: SX18-4 )
- Tryptone (Thermo Fisher Scientific, catalog number: B211705 )
- Yeast Extract (Thermo Fisher Scientific, catalog number: B288620 )
- Sodium chloride (NaCl) (Thermo Fisher Scientific, catalog number: BP358-212 )
- Potassium chloride (KCl) (Thermo Fisher Scientific, catalog number: BP366-500 )
- Sodium phosphate (Na2HPO4) (Thermo Fisher Scientific, catalog number: BP332-500 )
- Potassium phosphate (KH2PO4) (Thermo Fisher Scientific, catalog number: BP362-500 )
- Tris-Base (Thermo Fisher Scientific, catalog number: BP152-500 )
- 95% ethanol (Decon Labs, catalog number: 2805HC )
- Dry ice
- LB-Lennox (pH 6.5) (see Recipes)
- 10x PBS (see Recipes)
- 20 mM Tris-Base (pH 8.5) (see Recipes)
- 500 mM NaCl (see Recipes)
Equipment
- Shaker-capable of shaking at 200 rpm at 37 °C (VWR International, New Brunswick Scientific, model: Excella E25 )
- Water bath shaker-capable of shaking at 30 °C at 200 rpm (VWR International, New Brunswick Scientific, model: Classic C76 )
- LS6000IC liquid scintillation counting system (Beckman Coulter)
- Timer
- Autoclave
- Biomate 3S Spectrophotometer-capable of reading at OD600 (Thermo Fisher Scientific)
- Semimicro Cuvettes (Thermo Fisher Scientific, catalog number: 14-955-127 )
- Table top Centrifuge-capable of spinning at 15,000 rpm at 4 °C (Eppendorf, catalog number: 5424 )
- Micropipettes (1,000 μl, 200 μl, 20 μl)
- 250 ml Erlenmeyer flask
Procedure
文章信息
版权信息
© 2015 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Plecha, S. C. and Withey, J. H. (2015). [14C] Linoleic Acid Uptake and Fractionation Assay in Vibrio cholerae. Bio-protocol 5(24): e1682. DOI: 10.21769/BioProtoc.1682.
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分类
微生物学 > 微生物生物化学 > 脂质
微生物学 > 微生物细胞生物学 > 细胞器分离
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