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Preparation of Chloroplast Lipid Membrane and Lipid-protein Interaction Assay
叶绿体膜脂的制备及脂质-蛋白质相互作用的分析   

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Abstract

Lipid-Protein interaction assay is a method to search lipids, which are bound with proteins in vitro. Since membranes that are spotted with chloroplast lipids such as monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG), and sulfoquinovosyldiacylglycerol (SQDG) are not commercially available, we extracted these lipids from cyanobacterial cells and spotted them onto membranes. The prepared membranes could be used for lipid-protein interaction assay.

Keywords: Lipid-protein interaction assay(脂质-蛋白质相互作用分析), Lipid extraction(脂质提取), Chloroplast lipid(叶绿体膜脂)

Materials and Reagents

  1. Preparation of lipids for chloroplast lipid membrane
    1. Amersham Protran Premium NC0.2 (GE Healthcare, Dharmacon, catalog number: AP-10600081 )
    2. TLC silica gel 60 plate (Merck Millipore Corporation, catalog number: 105721 )
    3. Fused silica capillary column, 0.25 mm x 50 m (Shinwa Chemical Industries, catalog number: HR-SS-10 )
    4. Cells of the cyanobacterium, Synechocystis sp. PCC 6803 (for lipid extraction)
    5. Methanol (Wako Pure Chemical Industries, Siyaku, catalog number: 137-01823 )
    6. Chloroform (Wako Pure Chemical Industries, Siyaku, catalog number: 038-02601 )
    7. 0.01% primuline (Sigma-Aldrich, catalog number: 206865 ), in 80% acetone
    8. Pentadecanoic acid (Sigma-Aldrich, catalog number: 91446-5G )
    9. Methanolic HCl (Sigma-Aldrich, catalog number: 33050-U ), dilute with methanol 3 times
    10. Hexane (Wako Pure Chemical Industries, Siyaku, catalog number: 085-00411 )

  2. Lipid-Protein interaction assay
    1. PIP Strips, Membrane Lipid Strips (Echelon Biosciences, catalog numbers: P-6001 and P-6002 )
    2. Hybri-Bag (Cosmo Bio, catalog number: S-1001 )
    3. Albumin from bovine serum (BSA), fatty acid free (Wako Pure Chemical Industries, Siyaku, catalog number: 017-15146 )
    4. Purified tagged proteins
    5. An anti-penta-His mouse monoclonal antibody (QIAGEN, catalog number: 34660 ) and an anti-GST mouse monoclonal antibody (Sigma-Aldrich, catalog number: G1160 )
    6. Horseradish peroxidase (HRP)-conjugated goat anti-mouse secondary antibody (Thermo Fisher Scientific, catalog number: 31432 )
    7. HRP substrate solution (Thermo Fisher Scientific, Pierce, catalog number: NCI32132 )
    8. Phosphate (pH 7.4)
    9. NaCl
    10. 0.1 % Tween-20
    11. Phosphate buffered saline (PBS) (see Recipes)
    12. PBST (see Recipes)

Equipment

  1. Glass tubes (Iwaki, catalog numbers: 71-088-004 and 71-063-006 )
  2. Pasteur pipette (Sansyo, Iwaki, catalog number: IK-PAS-9P )
  3. Spectrophotometer (Scinteck Instruments, Pharmacia Biotech, model: Ultrospec 2000 )
  4. Vortex mixer
  5. Centrifuge (TOMY SEIKO CO., model: LC-120 )
  6. Vacuum dryer (Shimadzu Corporation, model: SPE-200 centrifuge evaporator )
  7. TLC developing chamber (AS ONE, catalog number: CK-0544-060 )
  8. Hair dryer (commercially available)
  9. UV illuminator
  10. Razor blade
  11. Heat block
  12. Gas chromatograph (Shimadzu Corporation, model: GC-18A )
  13. Fused silica capillary column, 0.25 mm x 50 m (Shinwa Chemical Industries, catalog number: HR-SS-10 )
  14. Data processor (Shimadzu Corporation, model: C-R2AX )

Procedure

  1. Preparation of chloroplast lipid membrane
    1. Collect cyanobacterial cells by centrifugation and suspend the collected cells in 2 ml of 0.9% KCl. 3 x 109 cells of Synechocystis PCC 6803 are sufficient [Number of cells were determined by spectrophotometer. Optical density at 730 nm (OD730) = 1.0 corresponds to 1 x 108 cells /ml for Synechocystis PCC 6803].
    2. Add 7.5 ml of methanol:chloroform (2:1, v/v) and vortex for 1 min.
    3. Add 2.5 ml of chloroform and 2.5 ml of H2O, and vortex.
    4. Centrifuge at 700 x g for 10 min. Collect lower phase, put it into clean tube and dry in a vacuum dryer. Measure the weight of the tube before use and after drying lipids. Calculate the yield of lipids.
    5. Dissolve dried lipids in chloroform at 10 mg/ml.
    6. Add developing solvent into a TLC developing chamber, close and leave it for 30 min as shown in Figure 1. Developing solvent is acetone:benzene:ultrapure water = 91:30:8 (v/v/v). Spot the chloroform solution containing 1-3 mg of lipids onto a TLC plate and place the plate into the TLC developing chamber.


      Figure 1. Separation of lipids on TLC plate. 1. Add developing solvent into a TLC developing chamber; 2. Spot lipid solution onto a TLC plate and place the plate into the chamber; 3. The TLC plate is developed in the chamber to separate lipids.

    7. When the solvent front has reached 1 cm from the top of the plate, remove the plate from the chamber and dry with cool wind provided by hair dryer.
    8. Spray 0.01% primuline in 80% acetone onto the plate. Lipids are viewed as spots under long wavelength of UV light as shown in Figure 2. Mark the corresponding position of each lipid and scrape the silica gel in the each spot from the TLC plate that is grass plate coated with silica gel. The silica gel can be scraped from the plate with a razor blade.


      Figure 2. Thin-layer chromatogram of cyanobacterial lipids visualized by UV. Total lipids extracted from cyanobacteria were developed in acetone:benzene:ultrapure water=91:30:8. 1. Pigments; 2. Monoglucosyldiacyalglycerol; 3. Monogalactosyldiacylglycerol (MGDG); 4. Digalactosyldiacylglycerol (DGDG); 5. Sulfoquinovosyldiacylglycerol (SQDG); 6. Phosphatidylglycerol (PG).

    9. Add 1 ml of chloroform to the silica gel scraped from the plate and vortex. Spin down the silica gel and transfer supernatant to a new tube. Repeat this extraction 3 times and evaporate chloroform in a vacuum dryer. Dissolve the obtained lipids in 100 μl of chloroform. The following 10 to 12 steps are required to determine the yield of obtained lipids.
    10. Take 5 μl of the chloroform solution containing each lipid to a new tube with a screw cap and add 50 nmol of pentadecanoic acid as an internal standard and 2.5 ml of 3% (w/v) methanolic HCl. Close the tube with the screw cap and incubate at 80 °C for 3 h. During the incubation fatty acids bound to lipids are derivatized to fatty acid methyl esters.
    11. After cooling the tube to room temperature, add 2.5 ml of hexane and vortex vigorously. Fatty acid methyl esters are extracted with hexane. Leave to stand for 5 min and collect upper phase (hexane phase), put it into a clean tube and evaporate hexane in a vacuum dryer.
    12. Dissolve the obtained fatty acid methyl esters in 50 μl of hexane and analyze them with a gas chromatograph. Temperatures for the injector, column and detector chambers were set at 250 °C, 170 °C and 250 °C, respectively. Determine lipid content by calculating total amount of fatty acid methyl esters based on the peak areas of fatty acid methyl esters in the obtained chromatograms. Pentadecanoic acid is not contained in cyanobacterial lipids, thus it can be used as an internal standard.
    13. Dilute lipids (the obtained lipids at the step A9) with chloroform to 0.1, 1 and 10 mg/ml.
    14. Spot the lipids corresponding to 0.1, 1 and 10 mg/ml in 1 μl of chloroform onto Amersham Protran Premium membrane and dry completely.

  2. Lipid-protein interaction assay
    1. Incubate the membrane with 5 ml of blocking solution (3% fatty acid-free BSA in PBS) for 1 h at room temperature with gentle agitation.
    2. Discard the blocking solution and incubate the membrane with 2 ml of blocking solution containing 0.5 μg/ml tagged-protein (His-tagged or GST-tagged protein) overnight at 4 °C. Put the membrane and solution into a Hybri-Bag and seal it.
    3. Discard the protein solution and wash the membrane with 10 ml of PBST three times at room temperature with gentle agitation for 10 min each.
    4. Discard the solution and incubate the membrane with 2 ml of blocking solution containing primary antibody (anti-penta-His mouse antibody 1:2,000 or anti-GST mouse monoclonal antibody 1:2,000) for 1 h at room temperature with gentle agitation. Put the membrane and solution into a Hybri-Bag and seal it.
    5. Discard the solution and wash the membrane with 10 ml of PBST three times at room temperature with gentle agitation for 10 min each.
    6. Discard the solution and incubate the membrane with 2 ml of blocking solution containing secondary antibody (HRP-conjugated goat anti-mouse antibody 1:20,000) for 1 h at room temperature with gentle agitation. Put the membrane and solution into a Hybri-Bag and seal it.
    7. Discard the solution and wash the membrane with 10 ml of PBST three times at room temperature with gentle agitation for 10 min each.
    8. Incubate the membrane with enough HRP substrate solution to cover the membrane (2 to 3 ml) at room temperature for 5 min.
    9. Wash the membrane with ultrapure water to stop the reaction and dry the membrane.
    10. Scan the membrane to record the obtained data.

Representative data


Figure 3. Representative results of Lipid-Protein Interaction Assay. Each spot on the left (PIP Strip) and middle membrane (Membrane Lipid Strip) contained 100 pmol of lipids. The right membrane contained chloroplast lipids, MGDG, DGDG, SQDG, and PG, and also contained cardiolipin (CL) as the control. The membranes were incubated with 0.5 μg/mL GST-PDV2. The abbreviations used are: LPA, lysophosphatidic acid; LPC, lysophosphatidylcholine; PI, phosphatidylinositol; PI3P, phosphatidylinositol 3-phosphate; PI4P, phosphatidylinositol 4-phosphate; PI5P, phosphatidylinositol 5-phosphate; PE, phosphatidylethanolamine; PC, phosphatidylcholine; PI(3, 4)P2, phosphatidylinositol 3, 4-bisphosphate; PI(3, 5)P2, phosphatidylinositol 3, 5-bisphosphate; PI(4, 5)P2, phosphatidylinositol 4, 5-bisphosphate; S1P, sphingosine 1-phosphate; PI(3, 4, 5)P3, phosphatidylinositol 3, 4, 5-triphosphate; PA, phosphatidic acid; PS, phosphatidylserine; TAG, triacylglycerol; DAG, 1, 2-diacylglycerol. The image was reprinted from Reference 2 with permission from The Plant Cell; copyright American Society of Plant Biologists (www.plantcell.org).

Recipes

  1. PBS
    10 mM phosphate (pH 7.4)
    150 mM NaCl
  2. PBST
    PBS
    0.1 % Tween-20

Acknowledgments

This protocol was adapted from the original work (Okazaki et al., 2015) to provide the detailed procedures. This work was supported by JSPS KAKENHI Grant Number 26840089.

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. Okazaki, K., Miyagishima, S. Y. and Wada, H. (2015). Phosphatidylinositol 4-phosphate negatively regulates chloroplast division in Arabidopsis. Plant Cell 27(3): 663-674.
  3. Sato, N. and Murata, N. (1988). Membrane lipids. Meth Enzymol 167: 251-259.

简介

脂质 - 蛋白质相互作用测定法是一种搜索在体外与蛋白质结合的脂质的方法。 由于用叶绿体脂质例如单半乳糖基二酰基甘油(MGDG),二半乳糖基二酰基甘油(DGDG)和磺基奎诺糖基二酰基甘油(SQDG)点样的膜不可商购,因此我们从蓝藻细胞中提取这些脂质并将它们点样到膜上。 所制备的膜可用于脂质 - 蛋白质相互作用测定。

关键字:脂质-蛋白质相互作用分析, 脂质提取, 叶绿体膜脂

材料和试剂

  1. 制备叶绿体脂质膜的脂质
    1. Amersham Protran Premium NC0.2(GE Healthcare,Dharmacon,目录号:AP-10600081)
    2. TLC硅胶60板(Merck Millipore Corporation,目录号:105721)
    3. 熔融硅石毛细管柱,0.25mm×50μm(Shinwa Chemical Industries,目录号:HR-SS-10)
    4. 蓝细菌的细胞,集胞藻。 PCC 6803(脂质提取)
    5. 甲醇(Wako Pure Chemical Industries,Siyaku,目录号:137-01823)
    6. 氯仿(Wako Pure Chemical Industries,Siyaku,目录号:038-02601)
    7. 0.01%甘菊糖(Sigma-Aldrich,目录号:206865)在80%丙酮中
    8. 十五烷酸(Sigma-Aldrich,目录号:91446-5G)
    9. 甲醇HCl(Sigma-Aldrich,目录号:33050-U),用甲醇稀释3次
    10. 己烷(Wako Pure Chemical Industries,Siyaku,目录号:085-00411)

  2. 脂质 - 蛋白质相互作用测定
    1. PIP条,膜脂质条(Echelon Biosciences,目录号:P-6001和P-6002)
    2. Hybri-Bag(Cosmo Bio,目录号:S-1001)
    3. 来自牛血清(BSA),不含脂肪酸的白蛋白(Wako Pure Chemical Industries,Siyaku,目录号:017-15146)
    4. 纯化的标签蛋白
    5. 将抗penta-His小鼠单克隆抗体(QIAGEN,目录号: ?34660)和抗GST小鼠单克隆抗体(Sigma-Aldrich, 目录号:G1160)
    6. 辣根过氧化物酶(HRP)结合 山羊抗小鼠二次抗体(Thermo Fisher Scientific,目录 号码:31432)
    7. HRP底物溶液(Thermo Fisher Scientific,Pierce,目录号:NCI32132)
    8. 磷酸盐(pH 7.4)
    9. NaCl
    10. 0.1%Tween-20
    11. 磷酸盐缓冲盐水(PBS)(见Recipes)
    12. PBST(参见配方)

设备

  1. 玻璃管(Iwaki,目录号:71-088-004和71-063-006)
  2. 巴斯德吸管(Sansyo,Iwaki,目录号:IK-PAS-9P)
  3. 分光光度计(Scinteck Instruments,Pharmacia Biotech,型号:Ultrospec 2000)
  4. 涡流搅拌器
  5. 离心机(TOMY SEIKO CO。,型号:LC-120)
  6. 真空干燥器(Shimadzu Corporation,型号:SPE-200离心蒸发器)
  7. TLC显影室(AS ONE,目录号:CK-0544-060)
  8. 吹风机(市售)
  9. 紫外照明灯
  10. 剃刀刀片
  11. 热块
  12. 气相色谱仪(Shimadzu Corporation,型号:GC-18A)
  13. 熔融硅石毛细管柱,0.25mm×50μm(Shinwa Chemical Industries,目录号:HR-SS-10)
  14. 数据处理器(Shimadzu Corporation,型号:C-R2AX)

程序

  1. 叶绿体脂质膜的制备
    1. 通过离心收集蓝细菌细胞并悬浮收集 ?细胞在2ml的0.9%KCl中。 3×10 9个细胞的集胞藻PCC 6803是 足够[通过分光光度计测定细胞数。 在730nm处的光密度(OD 730)= 1.0对应于1×10 8个细胞/ml ?为 Synechocystis PCC 6803]。
    2. 加入7.5ml甲醇:氯仿(2:1,v/v)并涡旋1分钟。
    3. 加入2.5ml氯仿和2.5ml H 2 O,并涡旋。
    4. 在700×g离心10分钟。收集下相,放入 清洁管并在真空干燥器中干燥。测量管的重量 使用前和干燥后脂质。计算脂质的产量。
    5. 将干燥的脂质溶解在氯仿中,浓度为10mg/ml。
    6. 将显影溶剂加入TLC显影室中,关闭 离开30分钟,如图1所示。显影溶剂为 丙酮:苯:超纯水= 91:30:8(v/v/v)。点氯仿 溶液包含1-3mg脂质到TLC板上, 板进入TLC显影室。


      图1.分离 脂质在TLC板上。1.将显影溶剂加入TLC显影液中 室; 2.将脂质溶液滴加到TLC板上并放置板 进入腔室; TLC板在室中显影 单独的脂质
    7. 当溶剂前沿达到1cm时 从板的顶部,从室中取出板并干燥 用吹风机提供的凉风。
    8. 喷雾0.01% 80%丙酮。脂质被视为长点下的斑点 波长的UV光如图2所示。标记相应的 位置并从每个斑点中刮掉硅胶 该TLC板是用硅胶包被的草板。硅胶 ?可以用刀片从刀片上刮下

      图2。 通过UV显现的蓝藻脂质的薄层色谱。 总计 ?从蓝藻中提取的脂质被开发 丙酮:苯:超纯水= 91:30:8。颜料; 2.单葡糖基二聚甘油; 3.单半乳糖基二酰基甘油(MGDG); 4。 半乳糖基二酰基甘油(DGDG); 5.磺基奎诺糖基二酰基甘油 (SQDG); 6.磷脂酰甘油(PG)。

    9. 加入1毫升 氯仿至从板上刮下的硅胶并涡旋。旋转 下移硅胶并将上清液转移到新管中。重复此操作 萃取3次,并在真空干燥器中蒸发氯仿。溶解 得到的脂质在100μl氯仿中。以下10?12 需要步骤来确定获得的脂质的产量
    10. 取5μl含有每种脂质的氯仿溶液到新管中 ?并加入50nmol的十五烷酸作为内部 标准品和2.5ml 3%(w/v)甲醇HCl。关闭管子 螺帽,并在80℃下孵育3小时。在孵化期间脂肪 与脂质结合的酸衍生为脂肪酸甲酯。
    11. 将管冷却至室温后,加入2.5ml己烷 ?大力涡旋。用己烷萃取脂肪酸甲酯。 静置5分钟,收集上相(己烷相),放入 并且在真空干燥器中蒸发己烷。
    12. 将获得的脂肪酸甲酯溶于50μl己烷中 用气相色谱仪分析它们。注射器的温度, 柱和检测器室设置在250℃,170℃和250℃, 分别。通过计算总量来确定脂质含量 基于脂肪酸甲酯的峰面积的脂肪酸甲酯 酯。十五酸不是 包含在蓝藻脂质中,因此它可以用作内部 标准。
    13. 用氯仿稀释脂质(在步骤A9获得的脂质)至0.1,1和10mg/ml。
    14. 点样脂质对应于0.1,1和10毫克/毫升在1微升 氯仿置于Amersham Protran Premium膜上并完全干燥。

  2. 脂质 - 蛋白质相互作用测定
    1. 孵育膜与5ml封闭溶液(3%无脂肪酸 ?BSA在PBS中)在室温下温和搅拌1小时。
    2. 弃去封闭溶液,孵育膜2毫升 包含0.5μg/ml标记蛋白(His-标记的或 GST标记的蛋白)在4℃过夜。把膜和溶液 进入Hybri袋并密封。
    3. 丢弃蛋白质溶液和 在室温下用10ml PBST洗涤膜三次 每次温和搅拌10分钟。
    4. 丢弃解决方案和 将膜与2ml含有一级的封闭溶液孵育 抗体(抗penta-His小鼠抗体1:2,000)或抗GST小鼠 单克隆抗体1:2000)在室温温育1小时 搅动。将膜和溶液放入Hybri袋中并密封
    5. 弃去溶液并用10ml PBST洗涤膜三次 次,在室温下温和搅拌10分钟。
    6. 弃去溶液,并用2ml封闭液孵育膜 溶液(HRP-缀合的山羊抗小鼠 抗体1:20000)在室温下温和搅拌1小时。 将膜和溶液放入Hybri袋中并密封
    7. 弃去溶液并用10ml PBST洗涤膜三次 次,在室温下温和搅拌10分钟。
    8. 孵育膜与足够的HRP底物溶液,以覆盖膜(2至3毫升)在室温下5分钟。
    9. 用超纯水洗涤膜以终止反应并干燥膜
    10. 扫描膜以记录所获得的数据。

代表数据


图3.脂质 - 蛋白质相互作用测定的代表性结果左侧(PIP条带)和中间膜(膜脂质条带)上的每个点含有100pmol脂质。右膜含有叶绿体脂质,MGDG,DGDG,SQDG和PG,并且还含有心磷脂(CL)作为对照。将膜与0.5μg/mL GST-PDV2一起温育。使用的缩写是:LPA,溶血磷脂酸; LPC,溶血磷脂酰胆碱; PI,磷脂酰肌醇; PI3P,磷脂酰肌醇3-磷酸; PI4P,磷脂酰肌醇4-磷酸; PI5P,磷脂酰肌醇5-磷酸; PE,磷脂酰乙醇胺; PC,磷脂酰胆碱; PI(3,4)P 2,磷脂酰肌醇3,4-二磷酸酯; PI(3,5)P 2,磷脂酰肌醇3,5-二磷酸酯; PI(4,5)P 2,磷脂酰肌醇4,5-二磷酸酯; S1P,鞘氨醇1-磷酸; PI(3,4,5)P 3,磷脂酰肌醇3,4,5-三磷酸; PA,磷脂酸; PS,磷脂酰丝氨酸; TAG,三酰基甘油; DAG,1,2-二酰基甘油。该图像从参考文献2重印,获得来自植物细胞的许可;版权所有美国植物生物学家协会(www.plantcell.org)。

食谱

  1. PBS
    10mM磷酸盐(pH7.4) 150mM NaCl
  2. PBST
    PBS
    0.1%Tween-20

致谢

该协议改编自原始工作(Okazaki等人,2015),以提供详细的程序。这项工作得到JSPS KAKENHI Grant号26840089的支持。

参考文献

  1. Bligh,E.G。和Dyer,W.J。(1959)。 总脂质提取和纯化的快速方法。 Can J Biochem Physiol 37(8):911-917
  2. Okazaki,K.,Miyagishima,S.Y.and Wada,H。(2015)。 磷脂酰肌醇4-磷酸负调节拟南芥中的叶绿体分裂。 植物细胞 27(3):663-674。
  3. Sato,N。和Murata,N。(1988)。 膜脂质 Meth Enzymol 167:251-259。< br />
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
引用:Okazaki, K., Miyagishima, S. and Wada, H. (2016). Preparation of Chloroplast Lipid Membrane and Lipid-protein Interaction Assay. Bio-protocol 6(3): e1720. DOI: 10.21769/BioProtoc.1720.
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