A Technique for the Measurement of in vitro Phospholipid Synthesis via Radioactive Labeling
采用放射性标记的体外磷脂合成测量法
发布: 2016年01月20日第6卷第2期 DOI: 10.21769/BioProtoc.1705 浏览次数: 13874
评审: Neelanjan BoseDaniel KrausAnonymous reviewer(s)
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参见作者原研究论文
The authors used this protocol in:
Aug 2014
Abstract
This is an assay designed to examine the radioactive phosphorous incorporation when the molecule is being synthesized, which means that only de novo synthesized phospholipids can be detected. Thus, with this technique it is possible to detect in vitro phospholipid synthesis under different required experimental conditions respect to controls (Guido and Caputto, 1990; Ferrero et al., 2014). There are different types of lipids. Among them we can find phospholipids, which contain glycerol esterified with two fatty acyl chains and a phosphate group that can also be bound to an organic molecule that acts as “hydrophilic head”, as shown in Figure 1 for the case of phosphatidylcholine. This structure confers amphipathic properties to lipid molecules that allow them to form lipid bilayers, making phospholipids the main components of biological membranes. 
Figure 1. Representation of phospholipid structure. Extracted from: http://bio1151.nicerweb.com/Locked/media/ch05/phospholipid.html
Radioactive (放射性)
In vitro (体外)
ATP-P32 (atp-p32)
Labeling (标记)
Materials and Reagents
- PYREX® 5 ml Rimless Kahn Culture Tubes (12 x 75 mm) (Corning, catalog number: 9820-12 )
- Scintillation vials (Sigma-Aldrich, catalog number: Z190527 )
- The enzymes and substrates are obtained as protein homogenates from homogenized cells or tissue (see Step 1 of the Procedure)
- Bio-Rad Protein Assay Dye Reagent Concentrate (Bio-Rad Laboratories, AbD Serotec®, catalog number: 5000006 )
- HEPES (Sigma-Aldrich, catalog number: H3375 )
- Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S7653 )
- Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P9333 )
- D-(+)-Glucose (Sigma-Aldrich, catalog number: G8270 )
- Magnesium chloride (MgCl2) (Sigma-Aldrich, catalog number: M8266 )
- [γ32P]ATP (PerkinElmer, catalog number: BLU002001MC )
- Chloroform (Sigma-Aldrich, catalog number: C2432 )
- Methanol (Sigma-Aldrich, catalog number: 34860 )
- Trichloroacetic acid (TCA) (Sigma-Aldrich, catalog number: T6399 )
- Phosphotungstic acid (PTA) (Sigma-Aldrich, catalog number: P4006 )
- Non-aqueous liquid scintillation cocktail (PerkinElmer, catalog number: 1200-434 )
- Buffer HEPES (see Recipes)
- Reaction buffer (see Recipes)
- TCA-PTA 10-1 (% w/v) (see Recipes)
- TCA-PTA 5-0.5 (% w/v) (see Recipes)
- Carrier brain homogenate (see Recipes)
Equipment
- Tip sonicator (Branson Sonic Power Company, model: Sonifier B-12 ) or ULTRA-TURRAX®
- Spectrophotometer (Wavelength to be used: 595 nm) (Shimadzu Scientific Instruments, model: BioSpec-mini )
- Vortex (IKA® VORTEX 3) (Sigma-Aldrich, catalog number: Z654779 )
- Pipettes (PIPETMAN® L Starter Kit) (Gilson, catalog number: F167350 )
- Centrifuge (Cavourargetina, model: VT-3216Dx24 )
- Water thermostatic bath (Vicking, model: Masson D )
- Scintillation counter (WinSpectral, Wallac, PerkinElmer)
- Laboratory prepared with all the required equipment for safe receiving, storage, manipulation and waste processing of radioactive material, according to laws governing in each country
- Geiger counter (Ludlum Measurements, model: 3 survey meter )
Procedure
文章信息
版权信息
© 2016 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Rodriguez-Berdini, L. and Ferrero, G. O. (2016). A Technique for the Measurement of in vitro Phospholipid Synthesis via Radioactive Labeling. Bio-protocol 6(2): e1705. DOI: 10.21769/BioProtoc.1705.
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分类
癌症生物学 > 通用技术 > 生物化学试验
生物化学 > 脂质 > 脂质测定
细胞生物学 > 细胞新陈代谢 > 脂质
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