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Lipid Extraction from HeLa Cells, Quantification of Lipids, Formation of Large Unilamellar Vesicles (LUVs) by Extrusion and in vitro Protein-lipid Binding Assays, Analysis of the Incubation Product by Transmission Electron Microscopy (TEM) and by Flotation across a Discontinuous Sucrose Gradient
以HeLa细胞研究蛋白质和膜相互作用的一系列方法:脂质提取和气相色谱定量;挤压法制备大单室脂质体(LUV);蛋白质和脂质体外孵育;采用透射电子显微镜(TM)和浮式离心法在不连续蔗糖梯度范围分析蛋白质和脂质互作;

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Abstract

Dissecting the interactions established between proteins and membranes in a given type of cells is not an easy task. Using a cell-free system of large unilamellar vesicles (LUVs) to analyze these interactions may help decipher these interactions and identify potential membrane deformations induced by the proteins incubated with these LUVs. This article describes the protocols for 1) extraction of total lipids from eukaryotic cells using the method developed by Bligh and Dyer (1959), 2) the quantification of glycerophospholipids by gas chromatography after methanolysis, followed by 3) the formation of LUVs by extrusion, 4) protein-lipid binding assay, 5) analysis of the incubation product by transmission electron microscopy (TEM) and by flotation across a discontinuous sucrose gradient and finally, 6) analysis of the proteins by immunoblot and revelation of the glycerophospholipids by iodin fumigation.

Keywords: Large Unilamellar Vesicles (LUVs)(大单层脂质体(爱)), Liposomes(脂质体), Protein-Lipid Binding Assay(蛋白质脂质结合试验), Discontinuous Sucrose Gradients(Discontinuous Sucrose梯度), Transmission Electron Microscopy(透射电子显微镜)

Background

Cell-free systems consisting in giant unilamellar vesicles (GUVs; vesicles composed of a single bilayer of phospholipids and with a diameter greater than 1 μm) or liposomes incubated with recombinant proteins may help understand these interactions. Depending on their diameter and number of lamellae, liposomes are classified into small unilamellar vesicles (SUVs; vesicles constituted of a single bilayer of phospholipids and with a diameter comprised between 20 and 100 nm), large unilamellar vesicles (LUVs; vesicles constituted of a single bilayer of phospholipids and with a diameter comprised between 100 and 400 nm), large multilamellar vesicles (MLVs; vesicles constituted of multiple phospholipid bilayers and with a diameter comprised between 200 nm and 3 μm) and multivesicular vesicles (MVVs; large vesicles composed of a single bilayer of phospholipids and containing several smaller vesicles, each composed of a single bilayer of phospholipids).
   When a dried mix of lipids is dispersed in an aqueous solvent, large multilamellar vesicles (LMVs) form spontaneously. Smaller liposomes (SUVs or LUVs) may then be formed by sonication or extrusion. Here we report the formation of LUVs by extrusion of LMVs formed from complex lipids extracted from HeLa cells and their use to investigate Toxoplasma proteins/membrane interactions by flotation across a sucrose gradient and by TEM.

Materials and Reagents

  1. PYREX® disposable glass conical centrifuge tubes without cap (capacity: 50 ml) (Sigma-Aldrich, catalog number: CLS9950250-72EA )
  2. Disposable screw thread culture tubes with marking spot (ø13 mm) (KIMAX test tubes with Teflon liner caps) (Thomas Scientic, catalog number: 9210J23 )
  3. Pasteur pipettes (non-plugged, L 5 ¾ in.) (Sigma-Aldrich, catalog number: S6018 )
  4. Polycarbonate membranes (Avanti Polar Lipids)
    Note: The diameter of their pores is defined by the manipulator (Example: 100 nm, Avanti Polar Lipids, catalog number: 610005 ).
  5. Grids for transmission electron microscopy (grid size 400 mesh x 62 μm pitch, copper) (Sigma Aldrich, catalog number: G5026 )
  6. 0.80 ml open ultra-clear centrifuge tubes (size 5 x 41 mm) (Beckman Coulter; catalog number: 344090 ) and split adaptors (Beckman Coulter; catalog number: 356860 )
  7. Nitrocellulose membranes for protein transfer as for example: nitrocellulose membranes, 0.2 µm, 8 x 12 cm (Thermo Fisher Scientific, catalog number: 77012 )
  8. HeLa cells (ATTC, catalog number: CCL-2 )
  9. Dulbecco’s modified Eagle’s medium (DMEM) (Thermo Fisher Scientific, catalog number: 41966-029/052 )
  10. Fetal bovine serum (FBS) (Eurobio, catalog number: CVFSVF0001 )
  11. Penicillin/streptomycin (PAN-Biotech, catalog number: P06-07100 )
  12. L-glutamine (200 mM) (Thermo Fisher Scientific, catalog number: 25030-024 )
  13. Dulbecco’s phosphate-buffered saline (DPBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 14190-094/069 )
  14. Chloroform (for HPLC, ≥ 99.8%, amylene stabilized) (Sigma-Aldrich, catalog number: 34854 )
  15. Methanol (for HPLC, ≥ 99.9%) (Sigma-Aldrich, catalog number: 34860 )
  16. Water sterile-filtered (BioReagent, suitable for cell culture) (Sigma-Aldrich, catalog number: W3500 )
  17. C21:0 fatty acid (Heneicosanoic acid) (Matreya, catalog number: 1241 )
  18. Hexane (anhydrous, 95%) (Sigma-Aldrich, catalog number: 296090 )
  19. Sulfuric acid (99.999%) (Sigma-Aldrich, catalog number: 339741 )
  20. HEPES free acid [N-(2-hydroxyethyl)piperazine-N’-(2-ethanesulfonic acid); 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid)] (Sigma-Aldrich, catalog number: H3375 )
  21. Sodium chloride (NaCl) (ACS reagent, ≥ 99.0%) (Sigma-Aldrich, catalog number: S9888 )
  22. Liquid N2
  23. Uranyl acetate (Electron Microscopy Sciences, catalog number: 22400 )
  24. Sucrose (≥ 99.5%) [α-D-Glucopyranosyl, β-D-fructofuranoside, D(+)-Saccharose] (Sigma-Aldrich, catalog number: S8501 )
  25. Iodine (ACS reagent, ≥ 99.8%, solid) (Sigma-Aldrich, catalog number: 207772 )
  26. Complete DMEM medium (see Recipes)
  27. Protein-lipid binding buffer (see Recipes)

Equipment

  1. Flasks (150 cm2) (Dominique Dutscher, catalog number: 190150 )
  2. 37 °C/5% CO2 cell culture incubator (Dominique Dutscher, catalog number: 911378M )
  3. Refrigerated bench centrifuge (Dominique Dutscher, catalog number: 472456 )
  4. Fume hood (standard equipment of any lab)
  5. Vortex (Dominique Dutscher, catalog number: 0 79030 )
  6. Bottle of nitrogen gas
  7. Freezer (-20 °C) (Dominique Dutscher, catalog number: 099288B )
  8. 10 μl positive displacement pipet such as microman gilson model M10 (Gilson, catalog number: F148501 )
  9. Oven (for dry heat: 100 °C) (Dominique Dutscher, catalog number: 780405 )
  10. BPX70 gas chromatography column (30 m x 0.25 mm ID BPX70 0.25 μm) (Trajan Scientific, catalog number: 0 54622 )
  11. Gas chromatography apparatus (Shimadzu, ref GC-2010 Plus High-end GC)
  12. Water bath (37 °C) (Dominique Dutscher, catalog number: 910648 )
  13. Extruder set with holder/heating block (Avanti Polar Lipids, catalog number: 610000 )
  14. 2 x 250 μl Hamilton® GASTIGHT® syringes, 1800 series (1825N, volume 250 μl, needle size 22s ga [bevel tip], needles L51 mm [2 in.]) (Sigma-Aldrich, catalog number: 21394 )
  15. Fridge (4 °C) (Dominique Dutscher, catalog number: 670251B )
  16. Rotating agitator (Dominique Dutscher, catalog number: 0 62646 )
  17. Carbon evaporator ( Q150T Turbo-Pumped sputter coater/carbon coater) (Quorum Technologies, model: Q150T)
  18. Electron transmission microscope such as JEOL-1400 plus (MET 120 kV) (JEOL, model: 1400 Plus )
  19. Ultracentrifuge type TL100 labtop equipped with a MLS-50 Swinging-Bucket rotor (Beckman Coulter, catalog number: 367280 )
  20. 12% Mini-PROTEAN® TGXTM precast protein gels (10-well, 30 µl) (Bio-Rad Laboratories, catalog number: 4561043 )
  21. Power supplier (Dominique Dutscher, catalog number: 0 49192 )
  22. SDS-PAGE apparatus (SDS-PAGE), as for example Mini-PROTEAN® Tetra vertical electrophoresis cell for mini precast gels, 2-gels (Bio-Rad Laboratories, catalog number: 1658005 )
  23. Protein transfer apparatus, as for example Trans-Blot® TurboTM transfer system (Bio-Rad Laboratories, catalog number: 1704150 )
  24. Thin layer chromatography plates: silica gel on TLC aluminium foils (silica gel matrix, L x W = 20 x 20 cm) (Sigma-Aldrich, catalog number: 60805 )
  25. Hermetic glass tank such as Aldrich® rectangular TLC developing tanks, complete (L x H x W = 27.0 x 26.5 x 7.0 cm) (Sigma-Aldrich, catalog number: Z126195 )

Procedure

  1. Extraction of total lipids from HeLa cells according to the protocol from Bligh and Dyer (1959)
    1. Plate 20 flasks of 150 cm2 with HeLa cells in complete DMEM medium and incubate them for 3 days at 37 °C, in presence of CO2 (up to 100% confluency).
    2. Rinse the monolayers with 20 ml of DPBS.
    3. Scrape the HeLa cells in DPBS and pool them in 50 ml DPBS in a 50 ml glass tube (PYREX® disposable glass conical centrifuge tubes without cap).
    4. Centrifuge the cells for 10 min, at 4 °C, 350 x g and get rid of the supernatant.
    5. In the fume hood, resuspend the cell pellet in 3 ml of chloroform/methanol (1:2) at room temperature.
    6. In the fume hood, vortex the cell lysate every 5 min for 30 min to solubilize the lipids (room temperature).
    7. In the fume hood, add 500 μl of chloroform and 900 μl of H2O, vortex.
    8. Centrifuge for 10 min at 1,300 x g, room temperature.
    9. In the fume hood, transfer the bottom organic phase into a new glass tube tube (Disposable screw thread culture tubes with marking spot, ø13 mm) (Figure 1).



      Figure 1. Photo of a tube showing the separation of the upper aqueous phase from the bottom organic phase

    10. In the fume hood, repeat the extraction with 500 μl of chloroform and 900 μl of H2O (steps A7-A9) to increase the lipid purity.
    11. In the fume hood, aliquot the bottom organic phase (1.5 ml per tube) into 5 ml glass tubes (disposable screw thread culture tubes).
    12. In the fume hood, evaporate the solvents under an N2 flux (0.5 to 1 psi).
      Note: This evaporation step also eliminates O2 molecules, which would oxidize the fatty acids of phospholipids.
    13. Keep the dried lipid films (~200 μg/tube) at -20 °C until being used.
      Note: Quantification of the glycerophospholipids is performed by gas chromatography after methanolysis (see next section).

  2. Quantification of extracted glycerophospholipids by gas chromatography after methanolysis
    1. To separate the fatty acids from the glycerol backbones and methylate them, a lipid film (such as the ones obtained at the end of section A) is resuspended in 3 ml of 2.5% H2SO4 (in methanol). Resuspend by pipetting several times. This step must be performed under a fume hood.
    2. Add a specific fatty acid as internal standard dispersed in methanol, for example 1 nmol (this should represent a small volume: usually 1 or 2 μl) of C21:0.
    3. Seal the tube hermetically with a teflon cap.
    4. The methylation of fatty acids is performed for 1 h at 100 °C in an oven.
    5. When the tube has cooled down (at room temperature: this takes just a few minutes), stop the reaction by adding 3 ml of H2O and mix by pipetting several times (in the fume hood).
    6. Phases are formed by the addition of 3 ml of hexane: mix by vortexing, centrifuge for 10 min at 1,300 x g, room temperature, and using a Pasteur pipette, transfer the upper phase containing the methylated fatty acids into a glass tube (disposable screw thread culture tube). This step must be performed under the fume hood.
    7. Repeat the hexane extraction on the aqueous bottom phase (step B6) and pool the second upper organic phase with the first one. This step must be performed under the fume hood.
    8. Dry the pooled hexane phases that contain the fatty acid methyl esters under a stream of N2 (0.5-1 psi). This step must be performed under the fume hood.
    9. The lipid film is resuspended in 10 μl hexane (in the fume hood), injected into a BPX70 gas chromatography column (SGE) and analyzed by gas chromatography using a flame ionization detector (Perkin Elmer). The used temperature program is 1) 7 min 30 sec at 130 °C, followed by 2) an increasing ramp of temperatures from 130 °C to 180 °C, with an increase of 3 °C per minute, and finally 10 min at 180 °C. The fatty acids are separated by the N2 mobile phase (3.5 ml/min) according to their size and solubility. They are quantified by comparison of their retention time with that of the C21:0 internal standard fatty acid that was added during the methanolysis step.
      Note: A 150 cm2 flask of confluent HeLa cells yields ~400-420 μg of glycerophospholipids.

  3. Formation of large unilamellar vesicles (LUVs) by extrusion
    1. Rehydrate a lipid film overnight at room temperature in 10 mM HEPES-150 mM NaCl (pH 7.4).
      Note: The lipids naturally organize themselves into MLVs of various diameters.
    2. To obtain a population of large unilamellar vesicles (LUVs) of defined diameter (example: 100 nm) the solution is:
      1. Frozen in liquid N2.
      2. Vortexed.
      3. Thawed in a 37 °C water bath.
        Note: The steps C2a-C2c, which are repeated 3 times, begin the separation of the lipid layers.
      4. Passed through an extruder: forcing 20 times the passage of MLVs through the calibrated pores of a polycarbonate membrane maintained between two Hamilton syringes separates the lipid bilayers one from another and allows the formation of LUVs. The photo of a mini-extruder equipped with two Hamilton syringes can be seen on the following website: https://avantilipids.com/divisions/equipment/mini-extruder-extrusion-technique/.
        Note: The LUV solution must be kept at 4 °C and be used for experiments within 2 days.

  4. Protein-lipid binding assay
    1. The binding assay is performed at room temperature for 30 min, under mild agitation (rotating agitator): 6 μg of purified (recombinant) protein are incubated with LUVs formed from 150 μg of extracted lipids and extruded at 100 nm.

  5. Analysis of the incubation product by TEM
    1. A carbon rod (3.05 mm diameter) is carved with a pencil sharpener.
    2. The carbon rod is placed between the two electrodes of the carbon evaporator and the mica sheet is positioned at the center of the evaporator chamber.
    3. Once the vacuum in the chamber is reached, carbon is evaporated for 400 msec.
    4. 2 μl of the incubation product are adsorbed on the clean side of a carbon film that had been pre-evaporated on a mica sheet.
    5. The carbon film is detached from the mica by floating it in a ceramic well (maximum volume: 200 μl) containing 100 to 150 μl of the negative stain solution (2% uranyl acetate solution) (Figure 2).


      Figure 2. Photo on how to float the carbon film into a well containing the negative stain solution

    6. The carbon film is picked up onto a 400 mesh copper grid using a small square of newspaper (Figure 3) and air dried.


      Figure 3. Photo illustrating how the copper grid covered by the carbon film is separated from the newspaper used to fish the grid from the well

    7. The grids are observed using a transmission electron microscope (TEM) such as a JEOL-1400 Plus (MET 120 kV) operating at 100 kV. Images are recorded at the nominal magnifications of 10,000x or 40,000x (see the section ‘Representative data’).

  6. Analysis of the incubation product by flotation across a discontinuous sucrose gradient
    1. The protein-lipid incubation product is mixed with 160 μl of a 40% sucrose solution and deposited at the bottom of a 750 μl open tube.
    2. Using a P200 micropipette, the bottom fraction is covered stepwise by 110 μl fractions of each of the following sucrose solutions: 30%, 20%, 10%, 5%, 0%.
      Note: The sucrose solutions are prepared in the binding buffer.
    3. The gradient is ultracentrifuged for 16 h, at 100,000 x g, 4 °C in a MLS-50 Swinging-Bucket rotor using a TL100 ultracentrifuge. The photo of a discontinuous sucrose gradient (although used for other purposes) can be observed on the Bio-Protocol ‘Isolation of Growth Cones from Mouse Brain’ (http://www.bio-protocol.org/e853, Figure 2, left image).
    4. The sucrose fractions are collected from the top and analyzed by
      1. Immunoblot to detect the studied protein (see section G below)
      2. Iodin fumigation to reveal the glycerophospholipids (see section H below)

  7. Analysis of the proteins by immunoblot
    1. 25 μl of each sucrose fraction (or 40 μl of the bottom sucrose fraction) are mixed with 5 μl of loading dye solution and loaded into an SDS-PAGE (percentage of polyacrylamide to be adjusted to the molecular weight of the studied protein, typically a 12% gel allows the resolution of proteins in the range of 15-250 kDa) (see http://www.bio-rad.com/webroot/web/pdf/lsr/literature/Bulletin_6040.pdf and BioProtocol [Bio101] Laemmli-SDS-PAGE).
    2. The proteins separated by SDS-PAGE are transferred onto a nitrocellulose membrane and further revealed using a specific antibody (standard immunoblot technique revealed by chemiluminescence [ECL]) (Figure 4A, lower panel).

  8. Revelation of the glycerophospholipids by iodin fumigation
    1. 5 μl of each sucrose fraction are spotted onto a Thin Layer Chromatography plate: use a pencil to draw a line and separate the spots one from each other by 1 cm on the line.
    2. The plate is introduced into a tank (a photo of such a tank can be seen on the website) saturated in iodin fumes (iodin crystals introduced into the tank 30 min before the introduction of the plate into the tank): iodin binds to the double bounds of fatty acids and thus allows to reveal the glycerophospholipids present in each fraction (Figure 4A, upper panel).

Representative data




Figure 4. Illustrations of two techniques commonly used to analyze the incubation product of liposomes and proteins. A. Analysis of the incubation product of the protein rGRA2 with HeLa LUV extruded at 100 nm. The protein was incubated with HeLa LUV at a lipid:protein ratio of 25:1. The binding mixture was mixed with 80% sucrose, loaded at the bottom of a tube, overlaid with a step sucrose gradient 40%-0% (bottom to top), and ultracentrifuged. Five microliters of each fraction were spotted onto nitrocellulose, and unsaturated phospholipids were revealed by fumigation with iodine (upper panel). An equal volume of each fraction was analyzed by western blot (WB) probed with anti-GRA2 mAb (lower panel). B. TEM analysis of the co-incubation of the proteins rGRA2 and rGRA6 with HeLa LUV extruded at 100 nm (lipid:protein, 50:1; rGRA2:rGRA6, 10:1). The right image represents a magnified view of the area in the white box. These figures are from the Figure 2 from Lopez et al. (2015).

Data analysis

The results depend on the composition in lipids of the LUVs and the nature of the protein incubated with them. The binding experiment as well as the flotation experiment/immunoblot analysis and the TEM observations should be repeated three times with similar results to guarantee the reproducibility of the results. The results are mainly qualitative. The relative amount of protein bound to LUV membranes and which thus floats up to the top fractions of the sucrose gradient might be quantified using the phosphorimager technology or by quantification of the signal pixels of the scanned ECL films using the ImageJ program (free software for Image Processing and Analysis in Java; https://imagej.nih.gov/ij/). The procedure used is the ‘Gel Analyzer’ method found as a submenu of the ‘Analyze’ menu (Tutorials and Examples: https://imagej.nih.gov/ij/docs/examples/index.html). Examples of such quantification can be found in Lopez et al. (2015).

Recipes

  1. Complete DMEM medium
    10% (v/v) FBS
    0.5% (v/v) penicillin/streptomycin
    2 mM glutamine
  2. Protein-lipid binding buffer
    10 mM HEPES (pH 7.4)
    150 mM NaCl

Acknowledgments

This protocol was adapted from: Lopez et al. (2015). This work was supported by a grant from Cluster 10 to Infectious Diseases- Rhône-Alpes region (C. Mercier); a PhD fellowship from the Rhône- Alpes region (A.B.). The authors thank Dr Eric Maréchal for the quantification of the extracted glycerophospholipids by gaz chromatography after methanolysis; Dr Pauline Ruffiot for the initial experiments of LUV formation, the former Master students Anne Valat, Khadi Sall, François Hermetet and Vincent Grassot for their participation to the project. No conflict or competing interest.

References

  1. Bligh, E. G. and Dyer, W. J. (1959). A rapid method of total lipid extraction and purification. Can J Biochem Physiol 37(8): 911-917.
  2. Lopez, J., Bittame, A., Massera, C., Vasseur, V., Effantin, G., Valat, A., Buaillon, C., Allart, S., Fox, B. A., Rommereim, L. M., Bzik, D. J., Schoehn, G., Weissenhorn, W., Dubremetz, J. F., Gagnon, J., Mercier, C., Cesbron-Delauw, M. F. and Blanchard, N. (2015). Intravacuolar membranes regulate CD8 T cell recognition of membrane-bound Toxoplasma gondii protective antigen. Cell Rep 13(10): 2273-2286.

简介

解剖在给定类型的细胞中蛋白质和膜之间建立的相互作用不是一个容易的任务。使用大单层囊泡(LUV)的无细胞系统来分析这些相互作用可以帮助破译这些相互作用和识别由与这些LUV孵育的蛋白质诱导的潜在的膜变形。本文介绍了1)使用由Bligh和Dyer(1959)开发的方法从真核细胞中提取总脂质,2)在甲醇分解后通过气相色谱法定量甘油磷脂,然后3)通过挤出形成LUV的方案, 4)蛋白质 - 脂质结合测定,5)通过透射电子显微镜(TEM)和通过不连续蔗糖梯度浮选分析孵育产物,最后,6)通过免疫印迹分析蛋白质并通过碘素熏蒸显示甘油磷脂。

[背景] 包含巨单层囊泡(GUV;由单个磷脂双层组成,直径大于1μm)或脂质体孵育的无细胞系统与重组蛋白可能有助于了解这些相互作用。根据它们的直径和层数,脂质体被分为小的单层囊泡(SUV;由单个磷脂双层构成的囊泡,直径在20和100nm之间),大的单层囊泡(LUV;由单个双层磷脂,并且直径在100和400nm之间),大多层囊泡(MLV;由多个磷脂双层构成且直径在200nm和3μm之间的囊泡)和多泡囊泡(MVV);由囊泡组成的大囊泡单个双层磷脂,并含有几个较小的囊泡,每个囊泡由单个双层磷脂组成)。  当脂质的干燥混合物分散在水性溶剂中时,大的多层囊泡(LMV)自发形成。然后可以通过超声处理或挤出形成较小的脂质体(SUV或LUV)。在这里我们报告挤压形成的LMVs从提取自HeLa细胞的复杂脂质的LUVs的形成和他们的使用通过浮选蔗糖梯度和TEM的调查弓形虫蛋白/膜相互作用。

关键字:大单层脂质体(爱), 脂质体, 蛋白质脂质结合试验, Discontinuous Sucrose梯度, 透射电子显微镜

材料和试剂

  1. (容量:50ml)(Sigma-Aldrich,目录号:CLS9950250-72EA)的一次性玻璃锥形离心管中加入
  2. 带有标记点(?13mm)(带特氟龙衬垫帽的KIMAX试管)(Thomas Scientic,目录号:9210J23)的一次性螺纹培养管
  3. 巴斯德移液管(非堵塞,L 5/4英寸)(Sigma-Aldrich,目录号:S6018)
  4. 聚碳酸酯膜(Avanti Polar Lipids)
    注意:它们的孔的直径由操纵器定义(例如:100nm,Avanti Polar Lipids,目录号:610005)。
  5. 用于透射电子显微镜(网格尺寸400目×62μm间距,铜)的栅格(Sigma Aldrich,目录号:G5026)
  6. (Beckman Coulter;目录号:344090)和分流适配器(Beckman Coulter;目录号:356860)的0.80ml开放超清离心管(尺寸5×41mm)
  7. 用于蛋白质转移的硝化纤维素膜,例如:硝酸纤维素膜,0.2μm,8×12cm(Thermo Fisher Scientific,目录号:77012)
  8. HeLa细胞(ATTC,目录号:CCL-2)
  9. Dulbecco改良的Eagle培养基(DMEM)(Thermo Fisher Scientific,目录号:41966-029/052)
  10. 胎牛血清(FBS)(Eurobio,目录号:CVFSVF0001)
  11. 青霉素/链霉素(PAN-Biotech,目录号:P06-07100)
  12. L-谷氨酰胺(200mM)(Thermo Fisher Scientific,目录号:25030-024)
  13. Dulbecco磷酸盐缓冲盐水(DPBS)(Thermo Fisher Scientific,Gibco TM,目录号:14190-094/069)
  14. 氯仿(HPLC,≥99.8%,亚苯基稳定)(Sigma-Aldrich,目录号:34854)
  15. 甲醇(对于HPLC,≥99.9%)(Sigma-Aldrich,目录号:34860)
  16. 水无菌过滤(BioReagent,适用于细胞培养)(Sigma-Aldrich,目录号:W3500)
  17. C21:0脂肪酸(二十一烷酸)(Matreya,目录号:1241)
  18. 己烷(无水,95%)(Sigma-Aldrich,目录号:296090)
  19. 硫酸(99.999%)(Sigma-Aldrich,目录号:339741)
  20. HEPES游离酸[N-(2-羟乙基)哌嗪-N' - (2-乙磺酸); 4-(2-羟乙基)-1-哌嗪乙磺酸]](Sigma-Aldrich,目录号:H3375)
  21. 氯化钠(NaCl)(ACS试剂,≥99.0%)(Sigma-Aldrich,目录号:S9888)
  22. 液体N <2>
  23. 乙酸乙酯(Electron Microscopy Sciences,目录号:22400)
  24. 蔗糖(≥99.5%)[α-D-吡喃葡萄糖基,β-D-呋喃果糖苷,D(+) - 蔗糖](Sigma-Aldrich,目录号:S8501)
  25. 碘(ACS试剂,≥99.8%,固体)(Sigma-Aldrich,目录号:207772)
  26. 完成DMEM培养基(参见配方)
  27. 蛋白质 - 脂质结合缓冲液(参见配方)

设备

  1. 烧瓶(150cm 2 )(Dominique Dutscher,目录号:190150)
  2. 37℃/5%CO 2细胞培养箱(Dominique Dutscher,目录号:911378M)中。
  3. 制冷台式离心机(Dominique Dutscher,目录号:472456)
  4. 通风橱(任何实验室的标准设备)
  5. Vortex(Dominique Dutscher,目录号:079030)
  6. 瓶氮气
  7. 冷冻器(-20℃)(Dominique Dutscher,目录号:099288B)
  8. 10μl正位移移液管如microman gilson模型M10(Gilson,目录号:F148501)
  9. 烤箱(干热:100℃)(Dominique Dutscher,目录号:780405)
  10. BPX70气相色谱柱(30m×0.25mm ID BPX700.25μm)(Trajan Scientific,目录号:054622)
  11. 气相色谱仪(Shimadzu,ref GC-2010 Plus High-end GC)
  12. 水浴(37℃)(Dominique Dutscher,目录号:910648)
  13. 带有夹持器/加热块的挤出机组(Avanti Polar Lipids,目录号:610000)
  14. 2×250μlHamilton注射器,1800系列(1825N,体积250μl,针头尺寸22s ga [斜角尖端],针头L51mm [2英寸] )(Sigma-Aldrich,目录号:21394)
  15. 冰箱(4℃)(Dominique Dutscher,目录号:670251B)
  16. 旋转搅拌器(Dominique Dutscher,目录号:062646)
  17. 碳蒸发器(Q150T Turbo-Pumped溅射涂布机/碳涂布机)(Quorum Technologies,型号:Q150T)
  18. 电子透射显微镜如JEOL-1400 plus(MET 120kV)(JEOL,型号:1400 Plus)
  19. 装备有MLS-50摆动转子转子(Beckman Coulter,目录号:367280)的超速离心机型TL100实验室顶部。
  20. 12%Mini-PROTEAN TGX 预制蛋白凝胶(10孔,30μl)(Bio-Rad Laboratories,目录号:4561043)
  21. 电源供应商(Dominique Dutscher,目录号:049192)
  22. SDS-PAGE装置(SDS-PAGE),例如用于微型预制凝胶的Mini-PROTEAN Tetra垂直电泳槽,2-凝胶(Bio-Rad Laboratories,目录号:1658005) >
  23. 蛋白转移装置,例如Trans-Blot TM Turbo TM sup/TM转移系统(Bio-Rad Laboratories,目录号:1704150)
  24. 薄层色谱板:TLC铝箔上的硅胶(硅胶基质,L×W = 20×20cm)(Sigma-Aldrich,目录号:60805)
  25. 密封玻璃罐如Aldrich(R)矩形TLC显影罐(L×H×W = 27.0×26.5×7.0cm)(Sigma-Aldrich,目录号:Z126195)

程序

  1. 根据来自Bligh和Dyer(1959)的方案从HeLa细胞中提取总脂质,
    1. 在完全DMEM培养基中用HeLa细胞平板培养150cm 2培养瓶,并在37℃,CO 2(高达100%汇合)存在下孵育3天)。
    2. 用20ml DPBS冲洗单层。
    3. 刮去DPBS中的HeLa细胞,并将它们在50ml玻璃管(PYREX 一次性玻璃锥形离心管,无帽)中的50ml DPBS中汇集。
    4. 离心细胞10分钟,在4℃,350×g ,并除去上清液。
    5. 在通风橱中,在室温下将细胞沉淀重悬在3ml氯仿/甲醇(1:2)中。
    6. 在通风橱中,每5分钟涡旋细胞裂解液30分钟以溶解脂质(室温)。
    7. 在通风橱中,加入500μl氯仿和900μlH 2 O,涡旋。
    8. 在室温下以1,300×g离心10分钟。
    9. 在通风橱中,将底部有机相转移到新的玻璃管管(带有标记点的一次性螺纹培养管,?13mm)(图1)。



      图1.显示上层水相与下层有机相分离的管的照片

    10. 在通风橱中,用500μl氯仿和900μlH 2 O O重复提取(步骤A7-A9)以提高脂质纯度。
    11. 在通风橱中,将底部有机相(每管1.5ml)等分到5ml玻璃管(一次性螺纹培养管)中。
    12. 在通风橱中,在N 2流量(0.5至1psi)下蒸发溶剂。
      注意:该蒸发步骤还消除O 2分子,其将氧化磷脂的脂肪酸。
    13. 保持干脂质膜(?200微克/管)在-20℃,直到使用 注意:在甲醇分解后,通过气相色谱法进行甘油磷脂的定量(参见下一部分)。

  2. 甲醇分解后用气相色谱法定量提取的甘油磷脂
    1. 为了从甘油骨架中分离脂肪酸并将其甲基化,将脂质膜(例如在部分A的末端获得的脂质膜)重悬浮在3ml的2.5%H 2 SO 4中, 4(在甲醇中)。通过移液器重悬几次。此步骤必须在通风橱下进行。
    2. 加入分散在甲醇中的特定脂肪酸作为内标,例如1nmol(这应该代表小体积:通常为1或2μl)的C21:0。
    3. 用聚四氟乙烯盖密封管子。
    4. 脂肪酸的甲基化在100℃下在烘箱中进行1小时
    5. 当管已经冷却(在室温:这只需要几分钟),通过加入3ml H 2 O停止反应,并通过吸移混合几次(在通风橱中)。
    6. 通过加入3ml己烷:混合物,通过涡旋,在室温下在1,300×g离心10分钟,形成相,使用巴斯德吸管,将含有甲基化脂肪酸的上层相转移到玻璃管(一次性螺纹培养管)。此步骤必须在通风橱下进行。
    7. 对含水底部相重复己烷萃取(步骤B6),并将第二上部有机相与第一个上部有机相合并。此步骤必须在通风橱下进行。
    8. 在N 2物流(0.5-1psi)下干燥含有脂肪酸甲酯的合并的己烷相。此步骤必须在通风橱下进行。
    9. 将脂质膜重悬于10μl己烷(通风橱)中,注入BPX70气相色谱柱(SGE)中,并使用火焰离子化检测器(Perkin Elmer)通过气相色谱法分析。使用的温度程序为:1)在130℃下7分30秒,然后2)温度从130℃至180℃的递增斜坡,以每分钟3℃的速率增加,最后在180℃下增加10分钟C。根据其大小和溶解度,通过N 2流动相(3.5ml/min)分离脂肪酸。通过将它们的保留时间与在甲醇分解步骤期间加入的C21:0内标脂肪酸的保留时间进行比较来量化它们。
      注意:150cm 2的合并的HeLa细胞烧瓶产生约400-420μg的甘油磷脂。

  3. 通过挤出形成大单层囊泡(LUV)
    1. 在室温下在10mM HEPES-150mM NaCl(pH 7.4)中将脂质膜再水合过夜 注意:脂质自然地组织成各种直径的MLV。
    2. 为了获得具有限定直径(例如:100nm)的大单层囊泡(LUV)群体,溶液是:
      1. 冷冻在液氮中。
      2. 旋涡。
      3. 在37℃水浴中解冻。
        注意:重复3次的步骤C2a-C2c开始分离脂质层。
      4. 通过挤出机:强制20倍MLV通过维持在两个Hamilton注射器之间的聚碳酸酯膜的校准孔,使脂质双层彼此分离,并允许形成LUV。装配有两个Hamilton注射器的小型挤出机的照片可以在以下网站上看到: https://avantilipids.com/divisions/equipment/mini-extruder-extrusion-technique/。
        注意:LUV溶液必须保持在4°C,并在2天内用于实验
  4. 蛋白质 - 脂质结合测定
    1. 结合测定在室温下在温和搅拌(旋转搅拌器)下进行30分钟:将6μg纯化(重组)蛋白与由150μg提取的脂质形成的LUV一起温育,并在100nm下挤出。
  5. 通过TEM分析孵育产物
    1. 用铅笔刀雕刻碳棒(直径3.05mm)。
    2. 碳棒放置在碳蒸发器的两个电极之间,云母片位于蒸发器室的中心。
    3. 一旦达到室中的真空,碳蒸发400毫秒
    4. 将2μl培养产物吸附在已预先蒸发在云母片上的碳膜的清洁侧上。
    5. 通过将碳膜漂浮在含有100-150μl阴性染色溶液(2%醋酸铀酰溶液)的陶瓷孔(最大体积:200μl)中(图2),使碳膜与云母分离。

      图2.关于如何将碳膜漂浮到含有负染色溶液的孔中的照片

    6. 使用小方形报纸(图3)将碳膜拾取到400目铜网上并空气干燥

      图3.照片说明了碳膜覆盖的铜网格如何与用于从井中捕获电网的报纸分离

    7. 使用透射电子显微镜(TEM)例如在100kV下操作的JEOL-1400 Plus(MET 120kV)观察栅格。图像以10,000x或40,000x的标称放大倍数记录(参见"代表数据"部分)
  6. 通过不连续蔗糖梯度浮选分析孵育产物
    1. 将蛋白质 - 脂质孵育产物与160μl40%蔗糖溶液混合并沉积在750μl开放管的底部。
    2. 使用P200微量吸移管,将底部馏分逐步用110μl级份的以下蔗糖溶液:30%,20%,10%,5%,0%覆盖。 注意:蔗糖溶液是在结合缓冲液中制备的。
    3. 在使用TL100超速离心机的MLS-50摆动转子中,将梯度超离心16小时,在100,000×g,4℃。不连续蔗糖梯度的照片(虽然用于其它目的)可以在Bio-Protocol"Isolation of Growth Cones from Mouse Brain"( http://www.bio-protocol.org/e853 ,图2,左图)。
    4. 从顶部收集蔗糖级分并通过
      分析
      1. 免疫印迹检测研究的蛋白质(见下文G部分)
      2. Iodin熏蒸显示甘油磷脂(见下面H部分)

  7. 通过免疫印迹分析蛋白质
    1. 将25μl每个蔗糖级分(或40μl底部蔗糖级分)与5μl加载染料溶液混合,并加载到SDS-PAGE(聚丙烯酰胺的百分比,以调节到所研究蛋白质的分子量,通常为12%凝胶允许分辨15-250kDa范围内的蛋白质(参见< a class ="ke-insertfile"href ="http://www.bio-rad.com/webroot/web/pdf/LSR /文学/Bulletin_6040.pdf"目标="_blank"> http://www.bio-rad.com/webroot/web/pdf/lsr/literature/Bulletin_6040.pdf 并BioProtocol [BIO 101] <一个class ="ke-insertfile"href ="http://www.bio-protocol.org/e80"target ="_ blank"> Laemmli-SDS-PAGE )。
    2. 将通过SDS-PAGE分离的蛋白质转移到硝酸纤维素膜上,并使用特异性抗体(通过化学发光显示的标准免疫印迹技术(ECL))进一步显示(图4A,下图)。
  8. 通过碘熏蒸引起甘油磷脂的显示
    1. 将5μl每种蔗糖部分点样到薄层色谱板上:使用铅笔画一条线,并在线上彼此分开1cm。
    2. 该板被引入到一个罐(例如一个槽的照片可以在<类="KE-的insertFile"可见HREF ="http://images.google.fr/imgres?imgurl=http%3A%2F %2Fwww.capitolscientific.com%2Fcore%2Fmedia%2Fmedia.nl%253Fid%253D96999%2526c%253D1250437%2526h%253Dbd0e36b51d03d1012532&imgrefurl =的http%3A%2F%2Fwww.capitolscientific.com%2FKONTES-416180-1020-长方矮个子薄-LAYER色谱 - 开发 - 坦克与盖为-10-X&H = 255&W = 255&tbnid = 2rkzc5Pia_YQ3M%3A和文档ID = 2HrttSP8M4QktM&EI = lHNyV4WcKsiQaJmVk_AL与TBM = isch与客户端= firefox的-b-AB&IACT = RC&uact = 3&DUR = 310页= 1&启动= 0&ndsp = 12&VED = 0ahUKEwiFtv6g4srNAhVICBoKHZnKBL4QMwggKAEwAQ&波黑= 481&BIW = 1089"目标="_空白">网站)在iodin烟雾(iodin引入引入板进入槽)之前的槽30分钟的晶体饱和:iodin结合的双键脂肪酸,因此允许揭示存在于每个级分中的甘油磷脂(图4A,上图)。

代表数据




图4.通常用于分析脂质体和蛋白质的孵育产物的两种技术的图示。A.蛋白质rGRA2与在100nm下挤出的HeLa LUV的孵育产物的分析。将蛋白质与脂质:蛋白质比例为25:1的HeLa LUV孵育。将结合混合物与80%蔗糖混合,装载在管的底部,用步骤蔗糖梯度40%-0%(从底部到顶部)覆盖,并超速离心。将5微升每种级分点在硝酸纤维素上,通过用碘熏蒸显示不饱和磷脂(上图)。通过用抗GRA2 mAb(下图)探测的蛋白质印迹(WB)分析等体积的各级分。 B.蛋白质rGRA2和rGRA6与在100nm下挤出的HeLa LUV(脂质:蛋白质,50:1; rGRA2:rGRA6,10:1)共孵育的TEM分析。右图像表示白框中的区域的放大视图。这些图来自Lopez等人的图2。 (2015)。

数据分析

结果取决于LUV的脂质中的组成和与它们孵育的蛋白质的性质。结合实验以及浮选实验/免疫印迹分析和TEM观察应该重复三次,具有类似的结果,以保证结果的重现性。结果主要是定性的。蛋白的相对量势必LUV膜和这因此漂浮到蔗糖梯度的顶部馏分可能使用磷光技术或通过使用ImageJ的程序(用于图像自由软件扫描ECL膜的信号象素的量化进行量化Java中的处理和分析; https://imagej.nih.gov/ij/)。使用的过程是"凝胶分析器"方法,作为"分析"菜单的子菜单(教程和示例: https://imagej.nih.gov/ij/docs/examples/index.html )。这种定量的实例可以在Lopez等人的中找到。 (2015)。

食谱

  1. 完成DMEM培养基
    10%(v/v)FBS
    0.5%(v/v)青霉素/链霉素 2mM谷氨酰胺
  2. 蛋白质 - 脂质结合缓冲液
    10mM HEPES(pH7.4) 150mM NaCl

致谢

该协议改编自:Lopez等人。 (2015)。这项工作得到了第10组传染病罗讷 - 阿尔卑斯地区(C. Mercier)的资助。来自罗纳 - 阿尔卑斯地区的博士研究生(A.B.)。作者感谢EricMaréchal博士在甲醇分解后通过gaz色谱法定量提取的甘油磷脂; Dr Pauline Ruffiot为LUV形成的初步实验,前硕士学生安妮Valat,Khadi Sall,Fran?oisHermetet和Vincent Grassot他们参与项目。没有冲突或竞争的利益。

参考文献

  1. Bligh,EG和Dyer,WJ(1959)。  A快速的总脂质提取和纯化方法。 Can J Biochem Physiol 37(8):911-917。
  2. Lopez,J.,Bittame,A.,Massera,C.,Vasseur,V.,Effantin,G.,Valat,A.,Buaillon,C.,Allart,S.,Fox,BA,Rommereim,LM,Bzik, DJ,Schoehn,G.,Weissenhorn,W.,Dubremetz,JF,Gagnon,J.,Mercier,C.,Cesbron-Delauw,MF和Blanchard,N。(2015)。  血管内膜调节膜结合的弓形虫保护性抗原的CD8T细胞识别。 Cell Rep 13(10):2273-2286。
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免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
引用:Bittame, A., Lopez, J., Effantin, G., Blanchard, N., Cesbron-Delauw, M., Gagnon, J. and Mercier, C. (2016). Lipid Extraction from HeLa Cells, Quantification of Lipids, Formation of Large Unilamellar Vesicles (LUVs) by Extrusion and in vitro Protein-lipid Binding Assays, Analysis of the Incubation Product by Transmission Electron Microscopy (TEM) and by Flotation across a Discontinuous Sucrose Gradient. Bio-protocol 6(20): e1963. DOI: 10.21769/BioProtoc.1963.
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