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In organello Protein Synthesis
细胞器的蛋白质合成   

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Abstract

In organello protein synthesis method allows the analysis of mitochondrial translation products. The principle of this method relies on incubation of isolated intact mitochondria with radiolabeled amino acids such as 35S methionine. After protein synthesis, the radiolabeled translation products are subsequently separated by SDS polyacrylamide gel electrophoresis and analysed by autoradiography. For in organello analysis of protein synthesis, the isolated intact mitochondria must retain their bioenergetics capacity, and in consequence be fully functional and able to perform coupled respiration. This in turn requires a quick and gentle purification of mitochondria during their isolation.

Keywords: In organello method(在organello方法), Protein synthesis(蛋白质的合成), Mitochondria(线粒体), Arabidopsis thaliana(拟南芥)

Materials and Reagents

  1. Youngest leaves harvested from 9- to 10-week-old Arabidopsis thaliana
  2. Common chemicals
    Klorin, NaCl, Tween-20, sucrose, tetrasodiumpyrophosphate, PVP-40, EDTA, KH2PO4, sodium ascorbate, L-cysteine, TES, BSA, GTP, mannitol, KCl, DTT, HEPES, MgCl2, sodium acetate, ADP, malic acid, pyruvate, puromycin, L-methionine, isopropanol, acetic acid, Commassie Blue G-250, glycerol, SDS, Tris, β-mercaptoethanol, bromophenol blue
  3. Sand (50-70 mesh particle size) (Sigma-Aldrich, catalog number: 274739 )
  4. Percoll (pH 8.5-9.5) (25 °C) (Sigma-Aldrich, catalog number: P1644 )
  5. L-35S methionine (HARTMANN ANALYTIC, catalog number: SRM-01H )
  6. Amino acid mixture without L-methionine (Promega Corporation, catalog number: L9961 )
  7. DC Protein Assay Kit (Bio-Rad Laboratories, catalog number: 500-0112 )
  8. 12% SDS-polyacrylamide gel (Leammli electrophoresis system)
  9. 10% chlorox solution (see Recipes)
  10. Grinding medium (see Recipes)
  11. 2x wash buffer (see Recipes)
  12. Synthesis mix (see Recipes)
  13. Stop solution (see Recipes)
  14. Isopropanol fixing solution (see Recipes)
  15. Rapid Coomassie Blue G-250 staining solution (see Recipes)
  16. Destaining solution (see Recipes)
  17. 1x solubilzation buffer (see Recipes)

Equipments

  1. Mortar
  2. Miracloth (Calbiochem®, catalog number: 475855-1R )
  3. Polycarbonate centrifuge tubes with round bottom (30 ml and 90 ml)
  4. 1.5 ml microcentrifuge tubes (SARSTEDT AG, catalog number: 72.690.001 )
  5. Paint brushes
  6. Tubes with round bottom (SARSTEDT AG, catalog number: 55.484.001 )
  7. Pasteur pipette
  8. Gradient former model 485 (Bio-Rad Laboratories, catalog number: 165-4120 )
  9. Peristaltic pump –PumpP-1 (GE Healthcare, catalog number: 18-111--91 )
  10. Centrifuges 1K15 and 3K18 (Sigma-Aldrich)
  11. Microcentrifuges (Eppendorf, catalog number: 5452 000.018 )
  12. Incubator shaker (IKA KS4000i control shaker, catalog number: 3510001 )
  13. Spectrophotometer UV-1800 (Schimadzu, catalog number: 206-25400-32 )
  14. SDS-PAGE system - Mini-Protean Tetra Cell (Bio-Rad Laboratories, catalog number: 165-8000 )
  15. Slab Gel dryer SGD5040 (Thermo Fisher Scientific, catalog number: SGD5040-230 )
  16. Radioactive room
  17. Carestream Kodak BioMax MR film (Sigma-Aldrich, catalog number: Z353949-50EA )

Procedure

  1. Isolation of mitochondria from leaves of Arabidopsis by a modification of the method described by Lister et al., (2007).
    Note: All subsequent steps perform at 4 °C in sterile conditions.
    1. Prepare 10% chlorox solution and store in cold room.
    2. Immediately prior to isolation, freshly add the following ingredients to previously prepared (see Recipe 2 below) Grinding medium and 2x wash buffer.
      Grinding medium                                     300 ml
      1% (w/v) BSA
      3.00 g
      18 mM sodium ascorbate
      1.06 g
      20 mM L-cysteine
      0.74 g
      2 x wash buffer
      150 ml
      0.2% (w/v) BSA
      0.30 g
      The remaining 50 ml of prepared 2x wash buffer (see Recipe 3 below) leave without BSA.
      Put these buffers in the cold room.
    3. Prepare heavy and light gradient solutions (35 ml). These recipes are sufficient for 4 gradient tubes.
      HEAVY (4.4% PVP-40)  
      2x wash buffer
      17.5 ml
      Percoll
      9.8 ml
      20% (w/v) PVP-40)
      7.7 ml
      LIGHT (0% PVP-40)      
      2x wash buffer
      17.5 ml
      Percoll
      9.8 ml
      MiliQ water
      7.7 ml
      Put these solutions in the cold room.
    4. Immediately after making a heavy and light gradient solutions prepare 1x wash buffer with BSA. For this purpose mix the residual volume of 2x wash buffer with BSA with the same volume of MiliQ water to obtain 1x wash buffer with BSA. Store this buffer in cold room.
    5. Weigh 25 g youngest leaves harvested from 9- to 10-week-old Arabidopsis thaliana growing under short-day conditions (SD, 22 °C) per sample.
    6. Sterilize leaves for 5 min in 10% cold chlorox solution. Then rinse the leaves 2-3 times for 1 min in cold sterile MiliQ water. Store leaves on ice, in dark.
      Note: Store leaves in dark avoid activation of photosynthesis inside chloroplasts (which can hamper the isolation of purified mitochondria).
    7. Put the leaves in 100 ml Grinding medium and cut the leaves with scissors. Then add 2.5 g of sterile sand (50-70 mesh particle size) and grind leaves energetically in a mortar for 30 sec.
    8. Filter homogenate through four layers of Miracloth into a conical flask.
    9. Transfer filtered homogenate (volume of Grinding medium + volume of grinding tissue) to 2 pre-chilled plastic centrifuge tubes (volume of tubes 90 ml).
    10. Centrifuge for 5 min at 2,450 x g at 4 °C → pellets cell debris and nuclei (general scheme of isolation mitochondria is presented in Figure 1).


      Figure 1. Scheme for the isolation of mitochondria from leaves of Arabidopsis thaliana

    11. Transfer supernatant to new tubes (volume of tubes 90 ml) and centrifuge for 12 min at 17,400 x g at 4 °C → pellets mitochondria, peroxisomes, etc.
    12. Discard supernatant and resuspend pellets in residual supernatant using a pre-wet with Grinding medium small paintbrush. Gently mix the residual supernatant touching with use of paintbrush the bottom of the tubes (volume of tubes 90 ml) to completely dissolve the pellet of mitochondria.
    13. Fill the tubes with 1x wash buffer with BSA and repeat centrifuge from steps 10-11 in order to pellet purified mitochondria.
    14. In the meantime make PVP-40 gradients in pre-chilled centrifuge tubes (volume of tubes 30 ml) using a gradient former model 485 (see Figure 2).


      Figure 2. Preparation of linear PVP-40 gradient with a gradient former. Set up tubes on ice and tape outflow tubes (TO) to the inside of the tubes. Close off the connection (TAP) between chambers. Pour 8,75 ml light gradient solution into chamber without tubing outlet. Pour 8,75 ml heavy gradient solution into chamber with tubing outlet and place a small magnetic stir bar (MSB) in this chamber, place gradient former on stirring block (SB). A magnetic stir bar should rotate at a speed sufficient to ensure complete mixing. Set peristaltic pump and allow for a moment only heavy solution to run. Open connection (TAP) between the chambers and allow solution to mix. It is important that the end of the outflow tube (TO) is held below the level of the gradient former so that solution will flow by gravity to down the side of a tubes held below the apparatus.

    15. After centrifugation from step 13 discard supernatant and resuspend pellets in 1.75 ml of 1x wash buffer with BSA using a small paintbrush. Combine the two resuspended pellets of the same type to one tube.
    16. Load sample carefully on PVP-40 gradient.
    17. Balance tubes and centrifuge for 20 min at 28,000 x g at 4 °C with the brakes off → separation of mitochondria from chloroplasts and thylakoids.
    18. After centrifugation mitochondria should form a light yellow/grey clouds at the bottom of the tubes. Carefully remove and discard the layer above the mitochondrial fraction by aspiration with a pasteur pipette.
    19. Collect mitochondrial fraction with use of truncated tip to a six 1.5 ml Eppendorf tubes filling with 1 ml of 1x wash buffer with BSA. To each tube collect 2 x 200 µl of mitochondrial fractions.
      Note: Use of truncated tips allow to collect dense and cloudy mitochondrial fractions.
    20. Centrifuge for 12 min at 21,000 x g at 4 °C.
    21. Remove supernatant carefully by pipette. Pellet might sit very loose.
    22. Resuspend gently pellets in all Eppendorf tubes in the remaining medium by snapping the tube. Combine the resuspended pellets of the same type to two arbitrarily chosen tubes.
    23. Fill these two tubes with 1x wash buffer without BSA and centrifuge for 12 min at 21,000 x g at 4 °C.
    24. Remove supernatant and resuspend pellets in the remaining medium by gently snapping the tube. Combine the two resuspended pellets of the same type to one tube.
      Note: The purpose of this step was purification of isolated mitochondria from Percoll. First the washes in more Eppendorf tubes allowed to discard the remaining contamination from gradients. In the next centrifugation steps the number of Eppendorf tubes were reduced to the one, containing purified mitochondria.
    25. Fill final tube with 1x wash buffer without BSA-centrifuge for 12 min at 21,000 x g at 4 °C.
    26. Remove supernatant and resuspended mitochondria in 30 µl of 1x wash buffer without BSA.
    27. Keep mitochondrial fraction on ice to use immediately in the in organello protein synthesis.

  2. Protein assay
    Determine the mitochondrial protein levels by DC Protein Assay Kit II, follow the instructions provided by the manufacturer. Estimate the protein concentrations for two independent amount of sample by comparison to a standard curve generated by the measurement of BSA of known concentrations. Using standard procedure, the assay is used with samples having protein concentrations between 0.2 and 1.5 mg/ml. The concentrations of mitochondrial proteins which we obtained are  between 20-25 µg/µl (mitochondrial fraction dissolved in 30 µl of 1x wash buffer).

  3. In organello protein synthesis
    For each mitochondrial preparation, set up two labeling reactions: one for the synthesis mix and one for control synthesis mix, to estimate the bacterial contamination. Use sodium acetate in control reactions instead of malic acid and pyruvate. Sodium acetate is a nonoxidizable substrate that can be utilized by bacteria but not by mitochondria, hence amino acid incorporation in its presence gives an indication of bacterial contamination.
    1. Prepare pre-chilled disposable, sterile 3.5 ml tubes with round bottom .
    2. Add 25 µl amino acids (without L-methionine) and 1 mg BSA to 1 ml of Synthesis Mix with malic acid and pyruvate or 1 ml of Control Synthesis Mix with sodium acetate. BSA protects the freshly isolated mitochondria. Keep on ice.
    3. Pipette to the pre-chilled tubes 100 µl of Synthesis Mix or 100 µl Control Synthesis Mix.
    4. Add to each tubes 150 µg of freshly isolated mitochondrial fractions.
    5. Add 30 µCi 35S L-methionine -1.5 µl (20 mCi/ml) per sample and incubate at 25 °C for 30 or 60 min in incubator shaker (speed 180 rpm).
      Note: During the assay keep the mitochondria well oxygenated using a shaker, to avoid the risk of anoxia due to their sedimentation to the bottom of the tube. In our experience, increasing the incubation times beyond 60 min does not increase the labeling of mitochondrial products.
    6. Following incubation reactions are stopped by addition 350 µl of ice cold 1x Wash buffer without BSA containing 10 mM unlabelled L-methionine and puromycin (50 µg/ml).
    7. Mix using pipette and transfer sample to new 1.5 ml Eppendorf tubes. Keep on ice.
    8. Pellet the mitochondrial fractions at 21,000 x g for 5 min in centrifuge at 4 °C.
    9. Remove the supernatant (containing the free 35S-methionine) and resuspend the mitochondrial pellet in 30 µl of 1x wash buffer without BSA.
    10. Mitochondria may be frozen on dry ice and stored at -80 °C or resuspended in 20-30 µl of electrophoresis sample buffer for immediate SDS-PAGE analysis (see D).

  4. SDS-Polyacrylamide gel electrophoresis and autoradiography
    1. Solubilize entire mitochondrial sample in a 1x solubilzation buffer  by heating for 5 min at 95 °C.
    2. Analyse the samples on 12% SDS-polyacrylamide gel (Leammli electrophoresis system).
    3. After electrophoresis gently agitate gel on a platform shaker in isopropanol fixing solution for 15 min.
    4. Discard fixing solution and gently agitate gel in Rapid Commasie Blue G-250 staining solution for 30 min.
    5. Remove staining solution and gently agitate gel in destaining solution for 40-60 min until a clear background with blue protein bands appears.
    6. Agitate gel in water 3 times for 5 min.
    7. After rinsing agitate gel in water containing 5% (v/v) of glycerol for 7 min. Agitation the gel in water containing glycerol may protect gel before cracking during drying.
    8. Dry gel onto a Whatman 3 MM paper using gel dryer, and expose for minimum 3 days to a Kodak BioMax MR film.

Representative data

  1. The example of autoradiogram of proteins synthesized in organello in Arabidopsis mitochondria is presented in below figure (Figure 3).


    Figure 3. In organello protein synthesis. Fragments of autoradiogram of proteins synthesized in organello for 30 and 60 min by wild-type mitochondria isolated from Arabidopsis (Kwasniak et al., 2013). After protein synthesis, 25 µg of mitochondria were fractionated on 12% (w/v) SDS-PAGE. The lane marked C. Bact. is a specific control performed in the presence of a sodium acetate- substrate for bacterial translation.

Recipes

Note: All the buffers and solutions are prepared with double-distilled water and sterilized by autoclave.

  1. 10% chlorox solution (1 L)
    Klorin          
    100 ml
    NaCl             
    10 g
    Tween-20     
    15 drops
  2. Grinding medium (300 ml)
    0.3 M sucrose
    30.80 g 
    25 mM tetrasodiumpyrophosphate (anhydrous)
    1.99 g
    2 mM EDTA (disodium salt)
    0.22 g
    10 mM KH2PO4
    0.40 g
    1% (w/v) PVP-40
    3.00 g
    Dissolve well in ~250 ml of MiliQ water, adjust pH to 7.5 with HCl and then make up to 300 ml with MiliQ water.
  3. 2x wash buffer (200 ml)
    0.6 M sucrose                                                      
    41.0 g
    20 mM TES                                                                
    0.9 g
    Dissolve well in ~150 ml of MiliQ water, adjust pH to 7.5 with NaOH and then make up to 200 ml with MiliQ water.
  4. Synthesis mix (20 ml)   
    5 mM KH2PO4
    13.61 mg
    2 mM GTPNa 
    20.93 mg
    0.4 M mannitol 
    1457.00 mg
    60 mM KCl   
    89.46 mg
    2 mM DTT  
    6.17 mg
    50 mM HEPES   
    238.31 mg
    10 mM MgCl2     
    40.66 mg
    4 mM ADP (K) 
    40.10 mg
    pH 7.0

    Mix solution and divide to
    1. 15 ml synthesis mix → add
      10 mM malic acid
      26.71 mg
      1 mM pyruvate (Na)
      1.65 mg
      Filter sterilize (0.22 µm pore size)
      15 x 1 ml
      Stored at -80 °C
    2. 5 ml control synthesis mix → add
      20 mM Na-sodium acetate
      8.20 mg
      Filter sterilize (0.22 µm pore size)
      5 x 1 ml
      Stored at -80 °C

  5. Stop solution (50 ml)
    10 mM L-methionine (0.0746 g) dissolved in 50 ml of 1x wash buffer without BSA
  6. Isopropanol fixing solution (1 L)
    Isopropanol      
    250 ml
    Acetic acid      
    100 ml
    Water               
    650 ml
  7. Rapid Coomassie Blue G-250 staining solution (1 L)
    Acetic acid         
    100 ml
    Water                                                
    900 ml
    Commassie brilliant blue G-250       
    60 mg
  8. Destaining solution (1 L)
    Acetic acid                                       
    100 ml
    Water                                                
    900 ml
  9. 1x solubilzation buffer
    2% (v/v) sodium dodecyl sulfate (SDS)
    10% glycerol
    62.5 mM Tris-HCl (pH 6.8)
    0.002 % (w/v) bromophenol blue
    5% β-mercaptoethanol
    Note: Indicated concentrations of components of solubilization buffer are final concentrations.

Acknowledgments

Functional mitochondria for in organello protein synthesis were isolated from leaves of Arabidopsis by a modification of the methods described by Lister et al., 2007 and Millar et al., 2007. In organello protein synthesis was performed for the first time on mitochondria isolated from leaves of Arabidopsis thaliana as described in the paper Kwasniak et al., 2013, and was based on previously published papers including Boutry et al., 1984. We are grateful to Christopher J. Leaver for several highly relevant papers that allow us to know details about the background of the in organello protein synthesis procedure.

References

  1. Boutry, M., Faber, A. M., Charbonnier, M. and Briquet, M. (1984). Microanalysis of plant mitochondrial protein synthesis products: Detection of variant polypeptides associated with cytoplasmic male sterility. Plant Mol Biol 3(6): 445-452.
  2. Echan, L. A. and Speicher, D. W. (2002). Protein detection in gels using fixation. Curr Protoc Protein Sci Chapter 10: Unit 10 15.
  3. Fernandez-Silva, P., Acin-Perez, R., Fernandez-Vizarra, E., Perez-Martos, A. and Enriquez, J. A. (2007). In vivo and in organello analyses of mitochondrial translation. Methods Cell Biol 80: 571-588.
  4. Grohmann, L. (1995). In organello protein synthesis. Methods Mol Biol 49: 391-397.
  5. Hakansson, G., van der Mark, F., Bonnett, H. T. and Glimelius, K. (1988). Variant mitochondrial protein and DNA patterns associated with cytoplasmic male-sterile lines of Nicotiana. Theor Appl Genet 76(3): 431-437.
  6. Kwasniak, M., Majewski, P., Skibior, R., Adamowicz, A., Czarna, M., Sliwinska, E. and Janska, H. (2013). Silencing of the nuclear RPS10 gene encoding mitochondrial ribosomal protein alters translation in arabidopsis mitochondria. Plant Cell 25(5): 1855-1867.
  7. Lister, R., Carrie, C., Duncan, O., Ho, L. H., Howell, K. A., Murcha, M. W. and Whelan, J. (2007). Functional definition of outer membrane proteins involved in preprotein import into mitochondria. Plant Cell 19(11): 3739-3759.
  8. Millar, A. H., Liddell, A. and Leaver, C. J. (2007). Isolation and subfractionation of mitochondria from plants. Methods Cell Biol 80: 65-90.

简介

在organello中,蛋白质合成方法允许分析线粒体翻译产物。 该方法的原理依赖于分离的完整线粒体与放射性标记的氨基酸如35 S-甲硫氨酸的孵育。 在蛋白质合成之后,随后通过SDS聚丙烯酰胺凝胶电泳分离放射性标记的翻译产物,并通过放射自显影进行分析。 对于蛋白质合成的分析,分离的完整线粒体必须保持其生物能量能力,因此是完全功能的并且能够进行耦合呼吸。 这反过来需要在其分离期间快速和温和地纯化线粒体。

关键字:在organello方法, 蛋白质的合成, 线粒体, 拟南芥

材料和试剂

  1. 从9至10周龄的拟南芥中获得的最嫩叶子
  2. 普通化学品
    Klorin,NaCl,吐温-20,蔗糖,焦磷酸四钠,PVP-40,EDTA,KH 2 PO 4,抗坏血酸钠,L-半胱氨酸,TES,BSA,GTP, 甘露醇,KCl,DTT,HEPES,MgCl 2,醋酸钠,ADP,苹果酸,丙酮酸盐,嘌呤霉素,L-甲硫氨酸,异丙醇,醋酸,Commassie Blue G-250,甘油, ,β-巯基乙醇,溴酚蓝
  3. 砂(50-70目粒径)(Sigma-Aldrich,目录号:274739)
  4. Percoll(pH8.5-9.5)(25℃)(Sigma-Aldrich,目录号:P1644)
  5. L-甲硫氨酸(HARTMANN ANALYTIC,目录号:SRM-01H)
  6. 不含L-甲硫氨酸的氨基酸混合物(Promega Corporation,目录号:L9961)
  7. DC蛋白测定试剂盒(Bio-Rad Laboratories,目录号:500-0112)
  8. 12%SDS-聚丙烯酰胺凝胶(Leammli电泳系统)
  9. 10%氯氧溶液(见配方)
  10. 研磨介质(见配方)
  11. 2x洗涤缓冲液(见配方)
  12. 合成混合(参见配方)
  13. 停止解决方案(参见配方)
  14. 异丙醇固定溶液(参见配方)
  15. 快速考马斯蓝G-250染色溶液(见配方)
  16. 解决方案(参见配方)
  17. 1x溶解缓冲液(见配方)

设备

  1. 砂浆
  2. Miracloth(Calbiochem ,目录号:475855-1R)
  3. 具有圆底的聚碳酸酯离心管(30ml和90ml)
  4. 1.5ml微量离心管(SARSTEDT AG,目录号:72.690.001)
  5. 油漆刷
  6. 圆底管(SARSTEDT AG,目录号:55.484.001)
  7. 巴斯德移液器
  8. 梯度成形器模型485(Bio-Rad Laboratories,目录号:165-4120)
  9. 蠕动泵-PumpP-1(GE Healthcare,目录号:18-111-91)
  10. 离心机1K15和3K18(Sigma-Aldrich)
  11. 微量离心机(Eppendorf,目录号:5452 000.018)
  12. 孵育振荡器(IKA KS4000i对照振荡器,目录号:3510001)
  13. 分光光度计UV-1800(Schimadzu,目录号:206-25400-32)
  14. SDS-PAGE系统 - Mini-Protean Tetra Cell(Bio-Rad Laboratories,目录号:165-8000)
  15. 板式凝胶干燥器SGD5040(Thermo Fisher Scientific,目录号:SGD5040-230)
  16. 放射性房间
  17. Carestream Kodak BioMax MR膜(Sigma-Aldrich,目录号:Z353949-50EA)

程序

  1. 通过Lister等人(2007)描述的方法的修改,从拟南芥的叶子中分离线粒体。</em> 注意:所有后续步骤在4°C在无菌条件下进行。
    1. 准备10%chlorox溶液,并储存在冷室。
    2. 在分离之前,立即加入以下成分 预先制备(见下面的配方2)研磨介质和2x洗涤 缓冲区 研磨介质                         ;             300ml
      1%(w/v)BSA
      3.00克
      18mM抗坏血酸钠
      1.06克
      20mM L-半胱氨酸 0.74克
      2×洗涤缓冲液
      150 ml
      0.2%(w/v)BSA 0.30 g
      剩余的50ml制备的2x洗涤缓冲液(见下面的配方3)不含BSA离开 将这些缓冲液放在冷室中。
    3. 制备重和轻梯度溶液(35ml)。 这些配方足以用于4个梯度管 HEAVY(4.4%PVP-40)  
      2x洗涤缓冲液
      17.5毫升
      Percoll
      9.8 ml
      20%(w/v)PVP-40) 7.7 ml
      LIGHT(0%PVP-40)      
      2x洗涤缓冲液
      17.5毫升
      Percoll
      9.8 ml
      MiliQ水
      7.7 ml
      将这些溶液放在冷室中。
    4. 在制备重和轻的梯度溶液后立即制备 1x含BSA的洗涤缓冲液。 为此目的混合2倍的残余体积 洗涤缓冲液用BSA与相同体积的MiliQ水混合,得到1x 洗涤缓冲液与BSA。 将此缓冲液储存在冷室中。
    5. 重量25   g在短日条件(SD,22℃)下每个样品生长的从9至10周龄的拟南芥中收获的最小的叶。
    6. 在10%冷chlorox溶液中灭菌叶片5分钟。 然后冲洗   叶片2-3次,在冷无菌MiliQ水中1分钟。 商店叶   冰,在黑暗中。
      注意:商店叶子在黑暗中避免激活 叶绿体内的光合作用(其可阻碍分离 纯化的线粒体)。
    7. 把叶子在100毫升研磨 介质和用剪刀切开叶子。 然后加入2.5g无菌砂 (50-70目粒度)并在研钵中大力研磨叶 30秒。
    8. 通过四层Miracloth过滤匀浆到锥形瓶中
    9. 转移过滤的匀浆(研磨介质的体积+ 研磨组织)至2个预先冷却的塑料离心管(体积 管90ml)。
    10. 在4℃下以2,450×g离心5分钟 沉淀细胞碎片和核(线粒体分离的一般方案   如图1所示)。


      图1.从拟南芥叶中分离线粒体的方案

    11. 转移上清液到新管(管的体积90毫升)和 在4℃下以17,400×g离心12分钟→粒线粒体, 过氧化物酶体,等。
    12. 弃去上清液并将沉淀重悬   残留上清液用预湿磨碎研磨介质小 画笔。 轻轻混合残留的上清液使用 画笔将试管的底部(试管体积90ml)完全   溶解线粒体的沉淀
    13. 用1x填充管 用BSA洗涤缓冲液,并从步骤10-11重复离心以便 颗粒纯化的线粒体
    14. 同时使PVP-40 梯度在预冷的离心管(管的体积30ml)中使用a   梯度模型485(见图2)

      图2.准备 的线性PVP-40梯度和梯度成形器。在冰上设置管 和流出管(TO)到管的内部。关闭 连接(TAP)。倒入8,75ml光梯度溶液 进入没有管出口的室。倒入8,75ml重梯度溶液  进入具有管出口的室中,并在该室中放置小的磁力搅拌棒(MSB),将梯度形成器置于搅拌块(SB)上。一个 磁力搅拌棒应以足以确保完成的速度旋转  混合。设置蠕动泵,并允许一瞬间只有重溶液  跑步。打开腔室之间的连接(TAP)并允许溶液 混合。重要的是,保持流出管(TO)的端部 低于梯度形成器的水平,使得溶液将流过 重力下降到保持在装置下方的管的侧面。

    15. 从步骤13离心后,弃去上清液并重悬 使用小画笔在1.75ml 1x BSA洗涤缓冲液中沉淀。 将两种相同类型的重悬浮颗粒合并到一个管中
    16. 在PVP-40梯度上小心装载样品。
    17. 平衡管并在4℃下以28,000×g离心20分钟   制动关闭→线粒体与叶绿体的分离 类囊体
    18. 离心后线粒体应形成 浅黄色/灰色云在管的底部。 小心取出 并通过抽吸丢弃线粒体组分上方的层 用巴斯德吸管。
    19. 收集线粒体级分 使用截短的尖端至填充1ml的六个1.5ml Eppendorf管 的1x洗涤缓冲液与BSA。 每管收集2×200μl 线粒体级分 注意:使用截短的提示允许收集密集和多云的线粒体部分。
    20. 在4℃下以21,000×g离心12分钟。
    21. 用移液管小心移除上清液。 颗粒可能坐得很松。
    22. 在剩余的所有Eppendorf管中轻轻地重悬 通过扣住管。 结合相同的重悬浮的丸粒   类型到两个任意选择的管。
    23. 用不含BSA的1x洗涤缓冲液填充这两个管,并在4℃下以21,000×g离心12分钟。
    24. 除去上清液,并在剩余培养基中重悬沉淀物 轻轻地扣住管子。 结合两个重悬的颗粒 相同类型到一个管 注意:此步骤的目的是 从Percoll纯化分离的线粒体。 首先洗涤 更多的Eppendorf管允许丢弃剩余的污染物   渐变。 在接下来的离心步骤中,Eppendorf的数量 管被还原为一个,含有纯化的线粒体。
    25. 在4℃下,在21,000×g下,用1×洗涤缓冲液(不含BSA-离心机)填充最终试管12分钟。
    26. 去除上清液和重悬的线粒体在30μl的1x无BSA洗涤缓冲液
    27. 保持线粒体部分在冰上立即用于有机物蛋白质合成中。
  2. 蛋白质测定
    通过DC蛋白测定试剂盒II确定线粒体蛋白水平,按照制造商提供的说明书。通过与通过测量已知浓度的BSA产生的标准曲线进行比较,估计两个独立量的样品的蛋白质浓度。使用标准程序,该测定用于蛋白质浓度在0.2和1.5mg/ml之间的样品。我们获得的线粒体蛋白的浓度是在20-25μg/μl(线粒体组分溶解在30μl的1x洗涤缓冲液中)之间
  3. 在有机物蛋白质合成中
    对于每个线粒体制剂,建立两个标记反应:一个用于合成混合物,一个用于对照合成混合物,以估计细菌污染。在对照反应中使用乙酸钠代替苹果酸和丙酮酸。乙酸钠是不可氧化的底物,其可以被细菌利用而不被线粒体利用,因此在其存在下的氨基酸掺入给出细菌污染的指示。
    1. 准备预冷的一次性无菌3.5 ml圆底管
    2. 添加25微升氨基酸(无L-甲硫氨酸)和1毫克BSA的1毫升 合成用苹果酸和丙酮酸或1ml对照合成混合 与乙酸钠混合。 BSA保护新鲜分离的线粒体。 保持冰上。
    3. 吸取预冷管100μl的合成混合物或100微升对照合成混合物
    4. 向每个管中加入150μg新鲜分离的线粒体级分
    5. 每个样品加入30μCi 35S L-甲硫氨酸-1.5μl(20mCi/ml)和 在25℃下在恒温振荡器(速度180rpm)中孵育30或60分钟 注意:   在测定期间,使用振荡器保持线粒体充分氧合, 以避免由于它们沉积到底部的缺氧的风险 管。 根据我们的经验,增加孵化时间超过60 min不增加线粒体产物的标记。
    6. 通过加入350μl冰终止孵育反应 冷1×含有10mM未标记L-甲硫氨酸的BSA的洗涤缓冲液   和嘌呤霉素(50μg/ml)
    7. 使用移液器混合,并将样品转移到新的1.5 ml Eppendorf管。 保持冰上。
    8. 在4℃下在离心机中将线粒体级分在21,000×g离心5分钟。
    9. 除去上清液(含有游离的35 S-甲硫氨酸)和 将线粒体沉淀重悬于30μl1x洗涤缓冲液中 BSA。
    10. 线粒体可以在干冰上冷冻并储存在-80℃ °C或重悬于20-30μl电泳样品缓冲液中 立即SDS-PAGE分析(见D)。

  4. SDS-聚丙烯酰胺凝胶电泳和放射自显影
    1. 将整个线粒体样品溶解于1x溶解缓冲液中 通过在95℃下加热5分钟。
    2. 在12%SDS-聚丙烯酰胺凝胶(Leammli电泳系统)上分析样品
    3. 电泳后,在平台振荡器上在异丙醇固定溶液中轻轻搅拌凝胶15分钟
    4. 弃去固定溶液,并在Rapid Commasie Blue G-250染色溶液中温和搅拌凝胶30分钟
    5. 去除染色溶液,轻轻搅拌脱色溶液中的凝胶   持续40-60分钟,直到出现具有蓝色蛋白条带的清晰背景
    6. 在水中搅拌凝胶3次,每次5分钟
    7. 在漂洗后,在含有5%(v/v)甘油的水中搅拌凝胶   7分钟。 在含有甘油的水中搅拌凝胶可以保护凝胶 在干燥期间开裂
    8. 使用凝胶干燥器将凝胶干燥到Whatman 3MM纸上,并且对Kodak BioMax MR膜曝光最少3天。

代表数据

  1. 在下图中显示了拟南芥线粒体中在有机物中合成的蛋白质的放射自显影图的实例(图3)。


    图3. 在有机体中蛋白质合成。在有机体中合成的蛋白质的放射自显影图片段在30和60分钟通过从< em> Arabidopsis (Kwasniak ,,2013)。 蛋白质合成后,在12%(w/v)SDS-PAGE上分离25μg线粒体。 标记为C. Bact的车道。 是在用于细菌翻译的乙酸钠 - 底物存在下进行的特异性对照

食谱

注意:所有缓冲液和溶液都用双蒸水制备,并通过高压灭菌器灭菌。

  1. 10%氯氧溶液(1L)
    Klorin          
    100 ml
    NaCl              
    10克
    Tween-20     
    15滴
  2. 研磨介质(300ml)
    0.3 M蔗糖 30.80克
    25mM焦磷酸四钠(无水)
    1.99克
    2mM EDTA(二钠盐) 0.22克
    10mM KH 2 PO 4 sub/
    0.40克
    1%(w/v)PVP-40
    3.00克
    充分溶解在〜250 ml MiliQ水中,用HCl调节pH至7.5,然后用MiliQ水补足至300 ml。
  3. 2x洗涤缓冲液(200ml)
    0.6 M蔗糖                                                    
    41.0克
    20mM TES                                                               
    0.9克
    充分溶解在〜150ml MiliQ水中,用NaOH调节pH至7.5,然后用MiliQ水补足至200ml。
  4. 合成混合物(20ml)  
    5mM KH 2 PO 4 sub/
    13.61 mg
    2 mM GTPNa
    20.93毫克
    0.4 M甘露醇
    1457.00 mg
    60 mM KCl   
    89.46毫克
    2 mM DTT  
    6.17 mg
    50 mM HEPES   
    238.31毫克
    10mM MgCl <2> 2      
    40.66mg
    4mM ADP(K)
    40.10 mg
    pH 7.0

    混合溶液并分配
    1. 15 ml合成混合→添加
      10mM苹果酸 26.71 mg
      1mM丙酮酸(Na) 1.65mg
      过滤灭菌(0.22μm孔径)
      15 x 1 ml
      储存于-80°C
    2. 5 ml对照合成混合→加入
      20mM Na-乙酸钠
      8.20 mg
      过滤灭菌(0.22μm孔径)
      5 x 1 ml
      储存于-80°C

  5. 停止溶液(50ml)
    10mM L-甲硫氨酸(0.0746g)溶于50ml不含BSA的1x洗涤缓冲液中
  6. 异丙醇固定液(1L)
    异丙醇       
    250 ml
    醋酸       
    100 ml
    水                
    650 ml
  7. 快速考马斯蓝G-250染色溶液(1L)
    乙酸          
    100 ml
    水水                                                
    900 ml
    Commassie亮蓝G-250       
    60 mg
  8. 脱色溶液(1 L)
    乙酸                          ;               
    100 ml
    水水                                                
    900 ml
  9. 1x溶解缓冲液
    2%(v/v)十二烷基硫酸钠(SDS)
    10%甘油 62.5mM Tris-HCl(pH6.8)
    0.002%(w/v)溴酚蓝
    5%β-巯基乙醇
    注意:溶解缓冲液成分的指示浓度为最终浓度。

致谢

通过修改Lister等,2007和Millar等所描述的方法从拟南芥的叶中分离用于有机物蛋白合成的功能性线粒体, 如在Kwasniak等人2013年的论文中所述,首次对从拟南芥叶子分离的线粒体进行有机物蛋白质合成,并且是基于以前发表的文章,包括Boutry 我们感谢Christopher J.Leaver关于几个高度相关的论文,其允许我们了解有机物蛋白质合成方法中的背景的细节。

参考文献

  1. Boutry,M.,Faber,A.M.,Charbonnier,M。和Briquet,M。(1984)。 植物线粒体蛋白质合成产物的微量分析:检测与细胞质雄性不育相关的变体多肽。 Plant Mol Biol 3(6):445-452。
  2. Echan,L.A。和Speicher,D.W。(2002)。 凝胶中的蛋白质检测使用固定。 Curr Protoc Protein Sci Chapter < em> 10:单位10 15.
  3. Fernandez-Silva,P.,Acin-Perez,R.,Fernandez-Vizarra,E.,Perez-Martos,A.and Enriquez,JA(2007)。体内 和在有机体中分析线粒体翻译。 Methods Cell Biol 80:571-588。
  4. Grohmann,L.(1995)。 在organello 蛋白质合成 Methods Mol Biol 49:391- 397。
  5. Hakansson,G.,van der Mark,F.,Bonnett,H.T.和Glimelius,K。(1988)。 与Nicotiana的细胞质雄性不育系相关的变体线粒体蛋白和DNA模式。 Theor Appl Genet 76(3):431-437。
  6. Kwasniak,M.,Majewski,P.,Skibior,R.,Adamowicz,A.,Czarna,M.,Sliwinska,E.and Janska,H。 沉默编码线粒体核糖体蛋白的核RPS10 基因改变了< em> arabidopsis mitochondria。 Plant Cell 25(5):1855-1867。
  7. Lister,R.,Carrie,C.,Duncan,O.,Ho,L.H.,Howell,K.A.,Murcha,M.W.and Whelan,J。(2007)。 涉及前蛋白导入线粒体的外膜蛋白的功能定义植物单元格 19(11):3739-3759。
  8. Millar,A.H.,Liddell,A.and Leaver,C.J。(2007)。 从植物中分离和亚分裂线粒体。 方法细胞生物学> 80:65-90。
  • English
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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。
Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
引用:Kwasniak-Owczarek, M. and Janska, H. (2014). In organello Protein Synthesis. Bio-protocol 4(12): e1157. DOI: 10.21769/BioProtoc.1157.
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Chris Leaver
University of Oxford
May be of interest:



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7/24/2014 9:42:44 AM Reply
Bio-protocol team Bio-protocol team
bio-protocol

Hi Chris, thank you very much for listing highly relevant papers that will allow our users to investigate the background of the protocol as well as extensive associated research. We would be particularly interested to hear back from you if you or somebody in your lab tries this protocol and shares their experience. We greatly value feedback from the community and strive to achieve maximum reproducibility of every protocol published on Bio-protocol.

7/28/2014 2:22:28 PM


Hanna Janska
Department of Biotechnology, University of Wroclaw, Poland

Hi Chris,

Thank you very much. I admire your work.

Regards, Hanna

8/1/2014 7:58:52 AM