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35S pulse Labelling of Chlamydomonas Chloroplast Proteins
衣藻属(Chlamydomonas )叶绿体蛋白质的35S脉冲标记   

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Abstract

35S pulse labelling of proteins is used to attach a radioactive label to newly synthesized proteins, as sulfur is an element that is mainly present in proteins (Fleischmann and Rochaix 1999). Depending on your organism’s uptake mechanisms you need cysteine, methionine or sulfuric acid as a source of radioactive sulfur. This example uses Chlamydomonas cells and H235SO4 (Schwarz et al., 2012).

Keywords: 35S pulse Labelling(35s脉冲标记), Chlamydomonas(衣藻), Protein Synthesis(蛋白质的合成), Chloroplast Proteins(叶绿体蛋白)

Materials and Reagents

  1. Strain of interest
  2. Control strains lacking the gene for the proteins of interest (as a negative control)
  3. Tris (Applichem, catalog number: A1379 )
  4. Ammonium chloride (Carl Roth, catalog number: 5470 )
  5. Magnesium chloride (Carl Roth, catalog number: KK36 )
  6. Calcium chloride (Merck, catalog number: 1023780500 )
  7. K2HPO4 (Applichem, catalog number: A1363 )
  8. KH2PO4 (Applichem, catalog number: A1364 )
  9. Na2EDTA (Carl Roth, catalog number: 8043 )
  10. ZnSO4·7H2O (Carl Roth, catalog number: T884 )
  11. H3BO3 (Carl Roth, catalog number: P010 )
  12. MnCl2·4H2O (Carl Roth, catalog number: 0 276 )
  13. FeSO4·7H2O (Carl Roth, catalog number: P015 )
  14. CoCl6H2O (Carl Roth, catalog number: T889 )
  15. CuSO4·5H2O (Carl Roth, catalog number: 8175 )
  16. (NH4)6Mo7O24·4H2O (Carl Roth, catalog number: 3666 )
  17. HEPES (Carl Roth, catalog number: HN78 )
  18. Tricine (Carl Roth, catalog number: 6977 )
  19. Methanol (Applichem, catalog number: A3493 )
  20. Cycloheximide (Carl Roth, catalog number: 8682 )
  21. 35S sulfuric acid (Hartmann Analytic, catalog number: S-RA-1 )
  22. Liquid nitrogen (Linde, inquire)
  23. Protease inhibitor cocktail (F. Hoffmann-La Roche, catalog number: 04693159001 )
  24. TAP-B (see Recipes)
  25. TAP-B/T(see Recipes)
  26. Hutner trace elements (see Recipes)
  27. Buffer A (see Recipes)
  28. Buffer B (see Recipes)

Equipment

  1. Sterile Erlenmeyer flasks (Brand KG)
  2. Photometer (GE Healthcare)
  3. Hemocytometer (Brand KG)
  4. Microscope (≥ 400x magnification, Leica)
  5. Reaction tubes (Sarstedt)
  6. Screw cap microreaction tubes (Sarstedt)
  7. Microreaction tube centrifuge with cooling capacity (Eppendorf)
  8. Gel dryer (Bio-Rad Laboratories)
  9. Whatman filter paper (GE Healthcare)

Procedure


I.  Sulfur deprivation

  1. Grow your strain of interest and the control strains at 23 °C to the early log phase (cell density < 2 x 106 cells/ml).
  2. Spin down your cells (5 min, 1,000 x g, RT) in sterile 50 ml reaction tube and resuspend carefully in 10 ml TAP-B, centrifuge again (5 min, 1,000 x g, RT) and resuspend the cells in 10 ml TAP-B.
  3. Transfer cells to a sterile 25 ml Erlenmeyer flask (it is best to set up the medium in this flasks and put the resuspended cells back in the same flask), shake for 16 h at 23 °C in medium light (30 – 50 μmol x m-2 x s-1).
  4. Spin down your cells (5 min, 1,000 x g, RT) and resuspend carefully in 10 ml TAP-B/T.
  5. Centrifuge again (5 min, 1,000 x g, RT) and resuspend the cells in exactly 10 ml TAP-B/T, transfer cells back to 25 ml Erlenmeyer flask.
  6. Agitate cells for 2 h in the dark (wrap flasks with aluminium foil or cover with cardboard box).


II. Adjustment of cells to the same amount of chlorophyll/cells

  1. Transfer 0.5 ml of the cultures to a microreaction tube (keep the remaining culture shaking in the dark).
  2. Centrifuge tube (2 min, 20,000 x g, 4 °C) and discard supernatant, resuspend pellet thoroughly in 1 ml methanol.
  3. Centrifuge again (1 min, 20,000 x g, 4 °C) and use supernatant for chlorophyll measurement at 652 nm, dilute with methanol (prechilling not necessary) if optical density is higher than 1 (do not forget to adjust your calculation for that dilution factor).
  4. Calculate chlorophyll content:



  5. Adjust Adjust chlorophyll content with TAP-B/T to 80 μg ml-1:



  6. Centrifuge cells (5 min, 1,000 x g, RT) and resuspend carefully in calculated volume of TAP-B/T to get 80 μg Chl/ml.
  7. If your cells are lacking chlorophyll better adjust the cell number than the chlorophyll amount, prepare a 1: 10 dilution of cultures and count the cells (hemocytometer, microscope), spin down cells and adjust cell number to 7.25 x 107 cells/ml (that corresponds to ~ 80 μg Chlorophyll/ml if using a green culture).


III. Pulse

  1. Add 25 μl of cycloheximide stock solution (100 μg/ml, final: 10 μg/ml) per culture to a screw cap tube (cycloheximide is for inhibition of cytosolic protein synthesis).
  2. Take your cultures and prepared tubes and go to fume hood in the radioactive lab (don’t inhale 35S! Release of radioactive gaseous SO2).
  3. Add 225 μl of each culture to the cycloheximide.
  4. Incubate for 10’ in rotary shaker in the dark (covered with cardboard box).
  5. Add 12.5 μl of 35S (H235SO4, 10 mCi/ml).
  6. Light pulse: incubate for 5-20 min in front of a appropriate light source and agitate cells occasionally.
  7. Spin down cells, discard supernatant (radioactive waste) and freeze the tube with the pellet in liquid nitrogen for at least 5 min to stop cellular activity.


IV. Cell lysis

  1. Thaw your cells on ice and keep them on ice from now on.
  2. Add 200 μl of buffer A and break the cells by pipetting up and down for ~ 1 min or sonication (three times 5 pulses at 50% output with 30’’ pauses in between).
  3. Remove soluble material: spin down membranes at 20,000 x g for 25 min at 4 °C, discard supernatant into radioactive waste.
  4. Resuspend membrane pellet with 100 μl of buffer B.


V.  Protein electrophoresis


Use a protocol for Laemmli-SDS-PAGE and adjust conditions for your protein depending on its molecular weight, e.g. a 6 M urea-16% polyacrylamide-SDS-PAGE gel for separation of the photosystem II reaction center proteins D1 (encoded by the psbA gene) and D2 (encoded by the psbD gene).


VI. Coomassie staining and drying of gel

  1. Use a protocol for Coomassie Blue staining (to visualize size marker and lanes).
  2. Put gel (upside down) on plastic tray, put Whatman paper (moistened with water) on top, flip over (Figure 1).
  3. Place Whatman paper and gel on top of two more layers of Whatman paper, cover with plastic foil and dry in gel dryer.


VII.Autoradiography

  1. Place phosphor imaging screen on top of your gel and expose for 1-3 days (or use X-ray film for longer time).
  2. Scan screen / develop X-ray film.
  3. Compare bands in your strains of interest with negative controls to identify affected protein, e.g. the band for the photosystem II reaction center protein D1 (encoded by the psbA gene) is missing in the investigated mutants as in the psbA mutant FuD7 in Figure 5 of (Morais et al., 1998).


    Figure 1. Scheme of step (VI-2).

Recipes

  1. TAP-B
    20 mM Tris
    7.5 mM ammonium chloride
    0.805 mM magnesium chloride
    0.34 mM calcium chloride
    0.537 mM K2HPO4
    0.463 mM KH2PO4
    0.1% Hutner trace elements
    Adjust to pH 7 with acetic acid
  2. TAP-B/T
    20 mM tris
    7.5 mM ammonium chloride
    0.805 mM magnesium chloride
    0.34 mM calcium chloride
    0.537 mM K2HPO4
    0.463 mM KH2PO4
    Adjust to pH 7 with acetic acid
  3. Hutner trace elements (Hill and Kafer, 2001)
    50 g Na2EDTA·2H2O
    22 g ZnSO7H2O
    11.4 g H3BO3
    5 g MnCl4H2O
    5 g FeSO4·7H2O
    1.6 g CoCl6H2O
    1.6 g CuSO4·5H2O
    1.1 g (NH4)6Mo7O24·4H2O
    Fill up with dH2O to 1,000 ml
  4. Buffer A
    10 mM EDTA
    10 mM HEPES (pH 7.8)
    Protease inhibitor cocktail (according to the manufacturer’s instructions)
  5. Buffer B
    10 mM EDTA
    10 mM tricine (pH 7.8)
    Protease inhibitor cocktail (according to the manufacturer’s instructions)

Acknowledgments

This protocol was adapted from the protocol published by Fleischmann and Rochaix (1999). The work was supported by a grant from the Deutsche Forschungsgemeinschaft to J.N. (grant number Ni390/4-2).

References

  1. Fleischmann, M. M. and Rochaix, J. D. (1999). Characterization of mutants with alterations of the phosphorylation site in the D2 photosystem II polypeptide of Chlamydomonas reinhardtii. Plant Physiol 119(4): 1557-1566.
  2. Hill T. W., Kafer E. (2001). Improved protocols for Aspergillus minimal medium: trace element and minimal medium salt stock solutions. Fungal Gen News 48: 20 -21
  3. Morais, F., Barber, J. and Nixon, P. J. (1998). The chloroplast-encoded alpha subunit of cytochrome b-559 is required for assembly of the photosystem two complex in both the light and the dark in Chlamydomonas reinhardtii. J Biol Chem 273(45): 29315-29320.
  4. Schwarz, C., Bohne, A. V., Wang, F., Cejudo, F. J. and Nickelsen, J. (2012). An intermolecular disulfide-based light switch for chloroplast psbD gene expression in Chlamydomonas reinhardtii. Plant J 72(3): 378-389.

简介

由于硫是主要存在于蛋白质中的元素(Fleischmann和Rochaix 1999),因此蛋白质的 S脉冲标记用于将放射性标记附着到新合成的蛋白质上。 根据你的生物体的吸收机制,你需要半胱氨酸,甲硫氨酸或硫酸作为放射性硫的来源。 该实施例使用衣原体细胞和H 2 35 SO Schwarz等人 。,2012)。

关键字:35s脉冲标记, 衣藻, 蛋白质的合成, 叶绿体蛋白

材料和试剂

  1. 感兴趣的
  2. 缺乏目标蛋白质基因的对照菌株(作为阴性对照)
  3. Tris(Applichem,目录号:A1379)
  4. 氯化铵(Carl Roth,目录号:5470)
  5. 氯化镁(Carl Roth,目录号:KK36)
  6. 氯化钙(Merck,目录号:1023780500)

  7. HPO 4(Applichem,目录号:A1363)

  8. (Applichem,目录号:A1364)
  9. Na 2 EDTA(Carl Roth,目录号:8043)
  10. ZnSO 4·7H 2 O(Carl Roth,目录号:T884)

  11. (Carl Roth,目录号:P010)
  12. MnCl 2·4H 2 O(Carl Roth,目录号:0276)
  13. FeSO 4 7HH 2 O(Carl Roth,目录号:P015)
  14. COCl 2·6H 2 O(Carl Roth,目录号:T889)
  15. CuSO 4 H 5·5H 2 O(Carl Roth,目录号:8175)
  16. (NH 4)6 Mo 6 Mo 7 O 24·4H 2 O(Carl Roth,目录号:3666)
  17. HEPES(Carl Roth,目录号:HN78)
  18. Tricine(Carl Roth,目录号:6977)
  19. 甲醇(Applichem,目录号:A3493)
  20. 环己酰亚胺(Carl Roth,目录号:8682)
  21. 硫酸(Hartmann Analytic,目录号:S-RA-1)
  22. 液氮(林德,查询)
  23. 蛋白酶抑制剂混合物(F.Hoffmann-La Roche,目录号:04693159001)
  24. TAP-B(见配方)
  25. TAP-B/T(参见配方)
  26. Hutner痕量元素(参见配方)
  27. 缓冲区A(参见配方)
  28. 缓冲液B(参见配方)

设备

  1. 灭菌锥瓶(Brand KG)
  2. 光度计(GE Healthcare)
  3. 血球计(品牌KG)
  4. 显微镜(放大倍数≥400x,徕卡)
  5. 反应管(Sarstedt)
  6. 螺旋盖微反应管(Sarstedt)
  7. 具有冷却能力的微反应管离心机(Eppendorf)
  8. 凝胶干燥器(Bio-Rad Laboratories)
  9. Whatman滤纸(GE Healthcare)

程序


I.  硫剥夺

  1. 在23℃下将感兴趣的菌株和对照菌株培养至早期对数期(细胞密度<2×10 6细胞/ml)。
  2. 在无菌的50ml反应管中旋转细胞(5分钟,1,000×g /孔,RT),并小心地重悬于10ml TAP-B中,再次离心(5分钟,1,000×g ,RT),并将细胞重悬于10ml TAP-B中
  3. 将细胞转移到无菌的25毫升锥形烧瓶中(最好是在这个烧瓶中设置培养基,并将重悬的细胞放回同一烧瓶中),在23℃下在中等光照下摇动16小时(30-50μmolxm -2 xs -1 )。
  4. 旋转你的细胞(5分钟,1000×g ,RT),并小心地重新悬浮在10ml TAP-B/T中。
  5. 再次离心(5分钟,1000×g/hr,RT),并将细胞重悬于精确的10ml TAP-B/T中,将细胞转移回25ml锥形瓶中。
  6. 在黑暗中搅动细胞2小时(用铝箔包裹烧瓶或用纸板盒盖)


II。 将细胞调节至相同量的叶绿素/细胞

  1. 转移0.5毫升培养物到微反应管(保持剩余的培养物在黑暗中摇动)
  2. 离心管(2分钟,20,000×g,4℃)并弃去上清液,将沉淀物在1ml甲醇中彻底重悬。
  3. 再次离心(1分钟,20,000xg,4℃),并使用上清液在652nm测量叶绿素,如果光密度高于1,则用甲醇稀释(不需要预冷)(不要忘记 调整稀释因子的计算)。
  4. 计算叶绿素含量:



  5. 调整用TAP-B/T将叶绿素含量调整为80μg/ml -1



  6. 离心细胞(5分钟,1,000×g /孔,RT),并在计算体积的TAP-B/T中仔细重悬,得到80μgChl/ml。
  7. 如果你的细胞缺少叶绿素,则比细胞数量更好地调节细胞数,制备1:10稀释的培养物并计数细胞(血细胞计数器,显微镜),旋转细胞并将细胞数调节到7.25×10 7,/sup>细胞/ml(如果使用绿色培养物,相当于〜80μg叶绿素/ml)


III。 脉冲

  1. 每个培养物加入25μl放线菌酮原液(100μg/ml,最终:10μg/ml)至螺旋盖管(放线菌酮用于抑制胞质蛋白合成)。
  2. 把你的文化和准备的管,并去通风橱在放射性实验室(不吸入 35 S释放放射性气体SO )。
  3. 将225微升的每种培养物加入放线菌酮中
  4. 在黑暗中的旋转振荡器(用纸板盒覆盖)中孵育10'
  5. 加入12.5μl的35 S(H 2 SO 4,SO 4 SO 4,10mCi/ml)。< br/>
  6. 光脉冲:在适当的光源前孵育5-20分钟,并偶尔搅拌细胞
  7. 旋转细胞,弃去上清液(放射性废物),并将沉淀物在液氮中冷冻至少5分钟以停止细胞活动。


IV。 细胞裂解

  1. 解冻你的细胞在冰上,并保持他们在冰上从现在开始
  2. 加入200μl缓冲液A,通过上下吹打约1分钟或超声处理破碎细胞(三次5次脉冲,50%输出,其间有30分钟的停顿)。
  3. 去除可溶性物质:在4℃下以20,000×g离心25分钟,将上清液丢弃为放射性废物。
  4. 用100μl缓冲液B重悬细胞沉淀


V.  蛋白电泳


使用用于Laemmli-SDS-PAGE的方案并根据其分子量调整蛋白质的条件,例如用于分离光系统II反应中心的6M尿素-16%聚丙烯酰胺-SDS-PAGE凝胶 蛋白D1(由psbA 基因编码)和D2(由psbD 基因编码)。


VI。 考马斯染色和凝胶干燥

  1. 使用考马斯蓝染色的协议(以显示大小标记和泳道)。
  2. 将凝胶(倒置)放在塑料托盘上,在顶部放置Whatman纸(用水润湿),翻转(图1)。
  3. 将Whatman纸和凝胶放在两层Whatman纸的顶部,用塑料箔覆盖并在凝胶干燥器中干燥。


七,自然放射学

  1. 将磷光体成像屏幕放置在凝胶顶部,暴露1-3天(或使用X光胶片较长时间)。
  2. 扫描屏/开发X光胶片
  3. 比较您感兴趣的菌株中的条带与阴性对照,以鉴定受影响的蛋白质,例如。 光系统II反应中心蛋白D1(由psbA 基因编码)的条带在 (Morais等人,1998年)的图5中的 psbA 突变体FuD7 中调查突变体。

    图1.步骤(VI-2)的方案。

食谱

  1. TAP-B
    20 mM Tris
    7.5mM氯化铵 0.805mM氯化镁 0.34mM氯化钙 0.537mM K 2 HPO 4
    0.463mM KH 2 PO 4>/
    0.1%Hutner痕量元素
    用乙酸调节pH至7
  2. TAP-B/T
    20mM tris
    7.5mM氯化铵 0.805mM氯化镁 0.34mM氯化钙 0.537mM K 2 HPO 4
    0.463mM KH 2 PO 4>/
    用乙酸调节pH至7
  3. Hutner微量元素(Hill和Kafer,2001)
    50g Na 2 EDTA·2H 2 O·h/v 22g ZnSO 4·7H 2 O x / 11.4g H sub 3 BO sub 3
    5g MnCl 2·4H 2 O·m/v 5g FeSO 4·7H 2 O·h/v 1.6克CoCl 2·6H 2 O 2·h/v 1.6克CuSO 4·5H 2 O 2 / 1.1g(NH 4)6 Mo 6 Mo 7 O 24·4H 2 O
    填充dH 2 2至1000ml
  4. 缓冲区A
    10 mM EDTA
    10mM HEPES(pH7.8)
    蛋白酶抑制剂混合物(根据制造商的说明书)
  5. 缓冲区B
    10 mM EDTA
    10mM三羟甲基氨基甲烷(pH7.8) 蛋白酶抑制剂混合物(根据制造商的说明书)

致谢

该协议改编自Fleischmann和Rochaix(1999)公布的方案。这项工作得到了德意志交响乐团对J.N. (授予号Ni390/4-2)。

参考文献

  1. Fleischmann,M.M.and Rochaix,J.D。(1999)。 表征具有莱茵衣藻D2光系统II多肽中磷酸化位点改变的突变体。 植物生理 119(4):1557-1566。
  2. Hill T.W.,Kafer E.(2001)。改进的曲霉属微生物培养基:微量元素和基本培养基盐溶液。真菌Gen新闻 48:20 -21
  3. Morais,F.,Barber,J.and Nixon,PJ(1998)。叶绿体编码α亚单位的细胞色素b-559是在莱茵衣藻光和黑暗中装配光系统两个复合物所必需的。 45):29315-29320。
  4. Schwarz,C.,Bohne,AV,Wang,F.,Cejudo,FJ和Nickelsen,J。(2012)。
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Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
引用:Bohne, A., Schwarz, C. and Nickelsen, J. (2013). 35S pulse Labelling of Chlamydomonas Chloroplast Proteins. Bio-protocol 3(11): e783. DOI: 10.21769/BioProtoc.783.
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