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Gradient Flotation Centrifugation of Chloroplast Membranes
叶绿体膜的梯度浮式离心实验   

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Abstract

Plastoglobules are lipoprotein particles physically attached to thylakoids in chloroplasts (Kessler et al., 1999). They are mainly composed of polar lipid, neutral lipids, and proteins (Vidi et al., 2006). Here we used simple sucrose gradient flotation centrifugation method to purify the plastoglobules from total chloroplast membranes (Vidi et al., 2007, Shanmugabalaji et al., 2013).

Keywords: Plastoglobule(plastoglobule), Gradient(梯度), Chloroplast(叶绿体), Membranes(膜), Centrifugation(离心的)

Materials and Reagents

  1. Anti-PGL35 (Agrisera, catalog number:  AS06 116 )
  2. Anti-TOC75 (Agrisera, catalog number:  AS06 150 )
  3. Anti-LHCB2 (Agrisera, catalog number:  AS01 003 )
  4. Na-ascorbate (Sigma-Aldrich, catalog number: 11140 )
  5. BSA fraction V (Sigma-Aldrich, catalog number: 05470 )
  6. PMSF/isopropanol (Sigma-Aldrich, catalog number: P7626 )
  7. Tricine-HCl (Sigma-Aldrich, catalog number: T0377 )
  8. DTT (Sigma-Aldrich, catalog number: 43819 )
  9. HB buffer (see Recipes)
  10. TE buffer (see Recipes)

Equipment

  1. Miracloth (pore size: 22-25 µm) (Merck KGaA, catalog number: 475855 )
  2. Centrifuge with JA-14 rotor
  3. Potter homogenizer
  4. Polycarbonate UltraClear SW28 tube
  5. SW41Ti rotor (Beckman Coulter)
  6. Spectrophotometer

Procedure

  1. Harvest leaves from of 3-4 weeks old Arabidopsis or tobacco seedlings (better if seedlings were kept in dark for 12 h before, to minimize the starch) and collect them in chilled water. Let them in cold room for at least 30 min.
  2. Grind leaves 3 times in 100 to 400 ml HB buffer, using a Waring blender homogeniser (1 time high, 5 sec; 3 times low, 3 sec).
  3. Filter the homogenate immediately through two cheese cloth and one miracloth.
  4. Centrifuge the filtrate 2 min at 2,200 rpm at 4 °C in JA-14 rotor.
  5. Resuspend the pellet in the 3 ml of HB buffer and quantify the chlorophyll.
  6. Adjust the volume to 50 ml with HB buffer and centrifuge at 4 °C at 3,600 rpm for 2 min.
  7. Resuspend the chloroplast pellet from the chloroplast prep in 0.6 M sucrose in TE buffer to a concentration of 1-2 mg/ml chlorophyll.
    Note: Chlorophyll content is measured by diluting 5-10 μl of resuspended chloroplasts into 1 ml of 80% acetone. Mix well and spin for 2 min in the microfuge. Remove the supernatant and measure against a blank of 80% acetone in a quartz cuvette at 652 nm.
    Chlorophyll concentration in mg/ml = OD652 x dilution factor/36
  8. Freeze the chloroplast suspension at -80 °C for 1-2 h.
  9. Thaw the suspension and dilute with at least 3 volumes of TE buffer.
  10. Homogenize for 20 strokes in a Potter homogenizer with a pestle.
  11. Spin the lysed chloroplasts at 39,000 rpm for 1 hour at 4 °C. Remove the brownish supernatant (stroma) by pipetting and store it in frozen at -20 °C for 30 min.
  12. Resuspend the pellet in 45% sucrose in TE buffer to a concentration of 2-3 mg chlorophyll/ml. Homogenize the pellet in a Potter homogenizer for 20 strokes as given above. The membranes may be stored at -20 °C at this point for later fractionation.
  13. Pipette the resuspended membranes into a polycarbonate UltraClear SW28 tube.
  14. Membranes were overlaid with a linear sucrose gradient created using 15 ml 5% sucrose and 15 ml of 45% sucrose in TE buffer and centrifuged for 17 h at 100,000 x g and 4 °C (SW41Ti rotor).
  15. Collect 1 ml of fractions from the gradient. The fractions can be stored at -20 °C.
    Starting from the top of the gradient (fraction 1) and ending at the bottom (approximately 32 fractions) plastoglobules are present in the fractions 1-6; envelopes are in fractions 14-18, and thylakoid membranes in fractions 25-32. Nevertheless, the exact chloroplast membrane distribution could be checked by immunoblotting with anti-PGL35 (plastoglobules), anti-TOC75 (envelopes) and anti-LHCB2 (thylakoid membranes).


    Figure 1. Purification of plastoglobules by flotation centrifugation. Total membranes from isolated chloroplasts were separated by flotation on a continuous sucrose gradient. Plastoglobules are visible as a yellowish green layer at the top of the gradient. THY, thylakoid membranes; PG, plastoglobules.

Recipes

  1. HB buffer

    Stock
    Final concentration
    For 400 ml
    For 200 ml
    Sorbitol (182.2  g/mol)
    450 mM
    32.8 g
    16.4 g
    1 M Tricine/KOH pH 8.4
    20 mM
    8 ml
    4 ml
    0.5 M EDTA  pH 8.5
    10 mM
    8 ml
    4 ml
    0.5 M NaHCO 3
    10 mM
    8 ml 
    4 ml
    1 M MnCl2
    1 mM
    0.4 ml
    0.2 ml
    Add the day of use:

    Na-ascorbate (200 g/mol)
    5 mM
    0.4 g
    0.2 g
    BSA fraction V
    0.05%
    0.2 g
    0.1 g
    0.2 M PMSF/isopropanol
    1 mM
    2 ml
    1 ml

  2. TE buffer

    Stock
    Final concentration
    Tricine-HCl (pH 7.5)
    50 mM
    EDTA
    2 mM
    DTT
    2 mM

Acknowledgments

This protocol was adapted from Shanmugabalaji et al. 2013. The project was supported by the NRP59, the University of Neuchatel, NCCR ‘‘Plant Survival’’ (National Center of Competence in Research), SystemsX PGCE and the Swiss National Foundation.

References

  1. Kessler, F., Schnell, D. and Blobel, G. (1999). Identification of proteins associated with plastoglobules isolated from pea (Pisum sativum L.) chloroplasts. Planta 208(1): 107-113.
  2. Vidi, P. A., Kanwischer, M., Baginsky, S., Austin, J. R., Csucs, G., Dormann, P., Kessler, F. and Brehelin, C. (2006). Tocopherol cyclase (VTE1) localization and vitamin E accumulation in chloroplast plastoglobule lipoprotein particles. J Biol Chem 281(16): 11225-11234.
  3. Vidi, P. A., Kessler, F. and Brehelin, C. (2007). Plastoglobules: a new address for targeting recombinant proteins in the chloroplast. BMC Biotechnol 7: 4. 
  4. Shanmugabalaji, V., Besagni, C., Piller, L. E., Douet, V., Ruf, S., Bock, R. and Kessler, F. (2013). Dual targeting of a mature plastoglobulin/fibrillin fusion protein to chloroplast plastoglobules and thylakoids in transplastomic tobacco plants. Plant Mol Biol 81(1-2): 13-25.

简介

塑性球蛋白是物理附着于叶绿体中类囊体的脂蛋白颗粒(Kessler等人,1999)。 它们主要由极性脂质,中性脂质和蛋白质组成(Vidi等人,2006)。 在这里,我们使用简单的蔗糖梯度浮选离心法从总叶绿体膜中纯化质体球(Vidi等人,2007,Shanmugabalaji等人,2013)。

关键字:plastoglobule, 梯度, 叶绿体, 膜, 离心的

材料和试剂

  1. 抗PGL35(Agrisera,目录号:AS06 116)
  2. Anti-TOC75(Agrisera,目录号:AS06 150)
  3. 抗-LHCB2(Agrisera,目录号:AS01 003)
  4. 抗坏血酸钠(Sigma-Aldrich,目录号:11140)
  5. BSA级分V(Sigma-Aldrich,目录号:05470)
  6. PMSF /异丙醇(Sigma-Aldrich,目录号:P7626)
  7. Tricine-HCl(Sigma-Aldrich,目录号:T0377)
  8. DTT(Sigma-Aldrich,目录号:43819)
  9. HB缓冲区(参见配方)
  10. TE缓冲区(参见配方)

设备

  1. Miracloth(孔径:22-25μm)(Merck KGaA,目录号:475855)
  2. 用JA-14转子离心机
  3. 波特均化器
  4. 聚碳酸酯UltraClear SW28管
  5. SW41Ti转子(Beckman Coulter)
  6. 分光光度计

程序

  1. 从3-4周龄的拟南芥或烟草幼苗收获叶子(如果幼苗在黑暗中保持12小时,以使淀粉最小化,则更好),并将其收集在冷水中。 让他们在寒冷的房间至少30分钟。
  2. 使用Waring搅拌器匀浆器(1次高,5秒; 3次低,3秒),在100至400ml HB缓冲液中研磨3次。
  3. 立即通过两个奶酪布和一个miracloth过滤匀浆
  4. 在4℃下,在JA-14转子中以2,200rpm离心滤液2分钟
  5. 将沉淀重悬于3ml HB缓冲液中,并定量叶绿素。
  6. 用HB缓冲液调节体积至50ml,并在4℃下以3,600rpm离心2分钟
  7. 将来自叶绿体制剂的0.6mM蔗糖的TE缓冲液中的叶绿体沉淀物重悬浮至1-2mg/ml叶绿素的浓度。
    注意:通过稀释5-10μl重悬浮来测量叶绿素含量 叶绿体置于1ml80%丙酮中。 充分混合并旋转2分钟 微量离心机。 除去上清液并对照空白的80%   丙酮在石英比色皿中在652nm。
    以mg/ml表示的叶绿素浓度= OD x稀释倍数/36
  8. 将叶绿体悬浮液在-80℃下冷冻1-2小时
  9. 解冻悬浮液并用至少3体积的TE缓冲液稀释
  10. 在带有杵的Potter匀浆器中匀浆20次
  11. 在4℃下以39,000rpm旋转裂解的叶绿体1小时。通过移液除去棕色的上清液(基质),并在-20°C冷冻储存30分钟
  12. 将沉淀物在45%蔗糖的TE缓冲液中重悬浮至2-3mg叶绿素/ml的浓度。在Potter匀浆器中将丸粒匀化20次,如上所述。此时可将膜在-20℃下储存以备以后分馏
  13. 将重悬的膜吸入聚碳酸酯UltraClear SW28管中。
  14. 将膜用由15ml 5%蔗糖和15ml 45%蔗糖的TE缓冲液制备的线性蔗糖梯度覆盖,并在100,000×g和4℃(SW41Ti转子)下离心17小时。 br />
  15. 从梯度中收集1ml级分。所述级分可储存在-20℃。
    从梯度的顶部(部分1)开始并在底部结束(大约32个部分),在部分1-6中存在增塑细胞;包膜在级分14-18中,类囊体膜在级分25-32中。 然而,可以通过用抗PGL35(塑料球),抗TOC75(包膜)和抗LHCB2(类囊体膜)的免疫印迹检查确切的叶绿体膜分布。


    图1.通过浮选离心纯化质体球。通过在连续蔗糖梯度上浮选分离来自分离的叶绿体的总膜。 在梯度的顶部,可以看到作为黄绿色层的塑料球。 THY,类囊体膜; PG,plastoglobules。

食谱

  1. HB缓冲区

    股票
    最终集中
    对于400 ml ,
    对于200 ml ,
    山梨醇(182.2g/mol)
    450 mM
    32.8克
    16.4克
    1 M Tricine/KOH pH 8.4
    20 mM
    8 ml
    4 ml
    0.5 M EDTA pH 8.5
    10 mM
    8 ml
    4 ml
    0.5 M NaHCO 3 3/h 10 mM
    8 ml 
    4ml
    1 M MnCl 2
    1 mM
    0.4 ml
    0.2 ml
    添加使用日期:

    抗坏血酸钠(200g/mol) 5 mM
    0.4克
    0.2 g
    BSA级分V
    0.05%
    0.2 g
    0.1 g
    0.2 M PMSF /异丙醇
    1 mM
    2 ml
    1ml

  2. TE缓冲区

    股票
    最终集中
    Tris-HCl(pH 7.5)
    50 mM
    EDTA
    2 mM
    DTT
    2mM

致谢

该方案由Shanmugabalaji等人修改2013年。该项目得到了NRP59,纳沙泰尔大学,NCCR'植物生存'(国家研究能力中心),SystemsX PGCE和 瑞士国家基金会。

参考文献

  1. Kessler,F。,Schnell,D。和Blobel,G。(1999)。 鉴定与豌豆分离的成纤维细胞相关的蛋白质(豌豆豌豆 )叶绿体。 Planta 208(1):107-113
  2. Vidi,P.A.,Kanwischer,M.,Baginsky,S.,Austin,J.R.,Csucs,G.,Dormann,P.,Kessler,F.and Brehelin,C.(2006)。 维生素E环化酶(VTE1)定位和维生素E在叶绿体plastoglobule脂蛋白颗粒中的积累。 J Biol Chem 281(16):11225-11234。
  3. Vidi,P.A.,Kessler,F。和Brehelin,C。(2007)。 Plastoglobules:一种用于在叶绿体中靶向重组蛋白的新地址。 BMC Biotechnol 7:4. 
  4. 这些研究结果表明,这些研究结果表明,这些研究结果表明,这些研究结果表明,这些研究结果表明, /www.ncbi.nlm.nih.gov/pubmed/23086498">双靶向成纤维细胞生长因子/纤丝蛋白融合蛋白与叶绿体平滑肌和类囊体在转基因烟草植物中的作用。 植物分子生物学 81(1-2):13-25。
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
引用:Shanmugabalaji, V. and Kessler, F. (2014). Gradient Flotation Centrifugation of Chloroplast Membranes. Bio-protocol 4(17): e1230. DOI: 10.21769/BioProtoc.1230.
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