Liposome Flotation Assays for Phosphoinositide-protein Interaction
磷酸肌醇-蛋白质相互作用的脂质体浮选分析法
发布: 2017年03月05日第7卷第5期 DOI: 10.21769/BioProtoc.2169 浏览次数: 18281
评审: Andrea PuharDaniel KrausVenkatasalam Shanmugabalaji
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参见作者原研究论文
The authors used this protocol in:
Feb 2015
Abstract
Phosphoinositides are rare membrane lipids involved in the control of the major cellular functions and signaling pathways. They are able to recruit specific effector proteins to the cytosolic face of plasma membrane and organelles to coordinate a vast variety of signaling and trafficking processes, as well to maintain specific identity of the different subcellular compartments (Di Paolo and De Camilli, 2006; Lemmon, 2003). Therefore, analysis of these effectors’ binding properties and specificity towards different phosphoinositides is crucial for the understanding of their cellular functions. This protocol describes a method to characterize the binding of proteins to different phosphoinositide-containing vesicles.
Keywords: Liposome flotation assay (脂质体浮选分析法)
Phosphoinositides (磷酸肌醇)
Protein-lipid interaction (蛋白质-脂质相互作用)
Unilamellar vesicles (单层囊泡)
Background
Several methods exist to analyze protein-phosphoinositide binding and the specificity towards the different members of the phosphoinositide family: lipid overlay, lipid flotation assay and surface plasma resonance (SPR). A Lipid flotation assay consists in the incubation of unilamellar vesicles with the protein of interest, and the subsequent flotation of the vesicles on a sucrose cushion, which will separate vesicle-bound proteins from unbound proteins and vesicles alone. Compared to the other methods a lipid flotation assay is i) technically easier and cheaper than SPR and ii) more specific and closer to physiological conditions, as it mimics the curvature of membranes, in contrast to protein-overlay assays, where lipids are dried on a flat nitrocellulose membrane. This protocol describes the binding of recombinant GST-tagged proteins to unilamellar vesicles of defined size and lipid composition, in particular regarding the specificity towards the different monophosphorylated phosphoinositides (PIP).
Materials and Reagents
- Glass tubes, 12 x 75 mm (DUTSCHER SCIENTIFIC, catalog number: 110011 )
- Tube, thickwall, polycarbonate, 1 ml, 11 x 34 mm (Beckman Coulter, catalog number: 343778 )
- Stericup-GP sterile vacuum filter unit, polyethersulfone, 0.22 μm (EMD Millipore, catalog number: SCGPU10RE )
- Mini Extruder Kit (mini-extruder, 2 Hamilton syringes, 0.1 μm polycarbonate membranes, filter supports, holder/heating block) (Avanti Polar Lipids, catalog number: 610000 )
- 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (PC) (Avanti Polar Lipids, catalog number: 850457 )
- 1-palmitoyl-3-oleoyl-sn-glycero-2-phosphoethanolamine (PE) (Echelon, catalog number: L-2368 )
- 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(carboxyfluorescein) (fluoPE) (Avanti Polar Lipids, catalog number: 810332 )
- Phosphatidylinositol 3-phosphate diC16 (PI3P) (Echelon, catalog number: P3016 )
- L-α-phosphatidylinositol-4-phosphate (PI4P) from porcine brain (Avanti Polar Lipids, catalog number: 840045 )
- Phosphatidylinositol 5-phosphate diC16 (PI5P) (Echelon, catalog number: P5016 )
- Liquid nitrogen
- Chloroform/methanol (50/50, v/v)
- PBS (w/o Ca/Mg) (Sigma-Aldrich, catalog number: D8537 )
- Sucrose (Sigma-Aldrich, catalog number: S0389 )
- Anti-GST antibody (clone B-14) (Santa Cruz Biotechnology, catalog number: sc-138 )
- Lipid stocks solutions (see Recipes)
- 60% and 25% sucrose solutions in PBS (see Recipes)
Equipment
- Nitrogen evaporator (N-EVAP, Organomation, catalog number: 11250 )
- Chemical hood
- Standard heated water bath
- Optima TLX ultracentrifuge (Beckman Coulter, model: OptimaTM TLX)
- TLA100.2 rotor (Beckman Coulter, model: TLA100.2 rotor)
Note: This rotor is not available anymore from Beckman Coulter. Rotor TLA120.2 (Beckman Coulter, model: TLA120.2) can be used instead. - Portable UV lamp
- SDS-PAGE running apparatus
- Western blotting apparatus
- Refractometer
Software
- Image analysis software (e.g., ImageJ, NIH)
Procedure
文章信息
版权信息
© 2017 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Tronchere, H. and Boal, F. (2017). Liposome Flotation Assays for Phosphoinositide-protein Interaction. Bio-protocol 7(5): e2169. DOI: 10.21769/BioProtoc.2169.
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分类
癌症生物学 > 通用技术 > 生物化学试验
细胞生物学 > 细胞信号传导 > 胞内信号传导
生物化学 > 脂质 > 脂质-蛋白互作
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