搜索

Investigating the Assembly Status of the Plastid Encoded Polymerase Using BN-PAGE and Sucrose Gradient Centrifugation
采用二维蓝绿温和胶聚丙烯酰胺凝胶(BN-PAGE)电泳和蔗糖梯度离心法研究质体中聚合酶的组装   

下载 PDF 引用 收藏 提问与回复 分享您的反馈 Cited by

本文章节

Abstract

The plastid encoded polymerase (PEP) represents a major transcription machinery in mature chloroplasts (Liere et al., 2011; Zhelyazkova et al., 2012). The proper assembly of this multi-subunit complex is important for plant growth and development (Pfalz and Pfannschmidt, 2013). The PEP polymerase can be purified from soluble and from membrane-bound (also named transcriptionally active chromosome, TAC) fractions. Blue Native polyacrylamide gel electrophoresis (BN-PAGE) and sucrose gradient sedimentation followed by immunoblot analyses is used to detect the status of the PEP complex assembly.

Keywords: Enrichment of the plastid encoded Polymerase (PEP)(Polymerase丰富的质体编码(PEP)), RNA Polymerase assembly(RNA聚合酶装配), Blue native PAGE (BN-PAGE)(蓝色变性PAGE(BN-PAGE)), Sucrose gradient centrifugation(蔗糖密度梯度离心法), Plastid protein complexes(质体蛋白复合物)

Materials and Reagents

  1. Material
    1. Disposable RNase-free pipette tips (1-200 µl and 100-1,000 µl universal tips)
    2. RNase-free microcentrifuge tubes (1.5 ml and 2.0 ml universal tubes)
    3. 15 ml conical tube
    4. Amicon Ultra-15 centrifugal filter units, 10KDa (Merck Millipore Corporation, catalog number: UFC901024 )
    5. PVDF-Membrane (Carl Roth, Roti® -Fluoro, catalog number: 2803.1 )
    6. Polypropylene centrifuge tubes, 14 x 95 mm (Beckman, catalog number: 331374 )

  2. Plant material
    1. Maize seedlings grown in soil for 7-10 days at 26-28 °C in cycles of 16 h-light/8 h-dark

  3. Antibodies
    1. anti-ZmpTAC12 (custom antibody) (Biogenes)
    2. anti-ZmRpoA (custom antibody) (Biogenes)
    3. Goat anti-Rabbit IgG HRP-linked (Sigma-Aldrich, catalog number: A6154 )

  4. Reagent
    1. Liquid nitrogen
    2. Acrylamide, Gel 40 (29:1) (Carl Roth, Rotiphorese®, catalog number: A515.1 )
    3. Ammonium persulfate (APS), (NH4)2S2O8 (Sigma-Aldrich, catalog number: 248614-500G-D )
    4. Bis-(2-hydroxyethyl)-imino-tris-(hydroxymethyl)-methane, Bis-Tris (Carl Roth, catalog number: 9140.3 )
    5. β-Mercaptoethanol (Carl Roth, catalog number: 4227.1 )
    6. Bradford, Roti®-Nanoquant (Carl Roth, catalog number: K880.1 )
    7. Coomassie brilliant blue G250 (Sigma-Aldrich, catalog number: B0770-5G )
    8. 1, 4-dithiothreitol, DTT (Carl Roth, catalog number: 6908.1 )
    9. ε-aminocaproic acid (Carl Roth, catalog number: 3113.2 )
    10. Glycerol (Carl Roth, catalog number: 6962.3 )
    11. Glycine (Carl Roth, catalog number: 3187.4 )
    12. HEPES (Carl Roth, catalog number: 9105.3 )
    13. Magnesium acetate tetrahydrate, Mg(CH3COO)2·4H2O, (Carl Roth, catalog number: 0 275.2 )
    14. Magnesium chloride, MgCl2 (Carl Roth, catalog number: KK36.3 )
    15. Methanol (Carl Roth, catalog number: P717.1 )
    16. n-Dodecyl β-D-maltoside, β-DM (Sigma-Aldrich, catalog number: D4641-1G )
    17. N, N, N’, N’-Tetramethylethylenediamine (TEMED) (Carl Roth, catalog number: 2367.1 )
    18. N-Tris-(hydroxymethyl)-methyl-glycin, Tricin (Carl Roth, catalog number: 6977.3 )
    19. BlueEasy Prestained Protein Marker (Nippon Genetics, catalog number: MWP06 )
    20. Potassium acetate, K(CH3COO) (Carl Roth, catalog number: T874.1 )
    21. Potassium hydroxide (Carl Roth, catalog number: 7986.1 )
    22. Protease inhibitor cocktail (cOmplete)
      Note: Currently, it is “Sigma-Aldrich, cOmpleteTM, catalog number: 000000011836153001 ”.
    23. Tris-(hydroxymethyl)-aminomethan, TRIS (Carl Roth, catalog number: 4855.3 )
    24. Triton X-100 (Carl Roth, catalog number: 3051.2 )
    25. Tween-20 (Carl Roth, catalog number: 9127.1 )
    26. Sodium chloride, NaCl (Carl Roth, catalog number: 9265.2 )
    27. Sodium dodecyl sulfate, SDS (Carl Roth, catalog number: 4360.2 )
    28. Sodium fluorid, NaF (Carl Roth, catalog number: 4503.2 )
    29. D-sucrose [D(+)-saccharose] Carl Roth, catalog number: 4621.2 )

  5. Buffers
    1. BN-Lysis buffer (see Recipes)
    2. 3x BN-Gel buffer (see Recipes)
    3. 4.5% Separating gel (see Recipes)
    4. 14% Separating gel (see Recipes)
    5. 4% Stacking gel (see Recipes)
    6. Cathode buffer blue (see Recipes)
    7. Cathode buffer (see Recipes)
    8. Anode buffer (see Recipes)
    9. 2x BisTris ACA (ACA, aminocaproic acid) (see Recipes)
    10. Sample buffer (see Recipes)
    11. 2% n-dodecyl-β-D-maltopyranoside (β-DM) solution (see Recipes)
    12. BN-Loading buffer (see Recipes)
    13. Transfer buffer (see Recipes)
    14. 1x TBST buffer (see Recipes)
    15. Low sucrose solution (see Recipes)
    16. High sucrose solution (see Recipes)
    17. Diluent solution (see Recipes)

Equipment

  1. Mortar and pestle
  2. Sonicator (BANDELIN electronic GmbH & Co., model: Sonopuls HD 2200 )
  3. Microcentrifuge (Beckman, model: Avanti JXN-30 )
  4. Ultracentrifuge (Sorvall Discovery 90SE)
    Note: This product has been discontinued by the manufacturer.
  5. SW40 rotor (Beckman)
  6. Gradient mixer (BIO-RAD, model: 385 )
    Note: This product has been discontinued by the manufacturer.
  7. BioPhotometer (Eppendorf)
  8. Electrophoresis apparatus (Hoefer, model: SE600 )
  9. Semi-Dry-Blotter (VWR International, PEQLAB Biotechnologie GmbH)

Procedure

  1. Blue Native polyacrylamide gel electrophoresis (BN-PAGE) analysis
    1. Collect plant tissue (basal half of one second leaf) into a 1.5 ml microcentrifuge tube and quickly freeze in liquid nitrogen. Frozen tissues can be stored at -80 °C for 1-2 months.
      Note: Since the expression of PEP subunits is higher in younger than in mature chloroplasts, proteins from young leaf sections (basal half of second leaves) were used in this study.
    2. Grind tissue in pre-chilled mortar with a pestle to a fine powder in liquid nitrogen.
    3. Let all liquid nitrogen evaporate and then immediately add 200-400 µl ice-cold BN-Lysis buffer directly to the tissue in the mortar. Do not let the tissue thaw before you add the BN-Lysis buffer.
    4. If the lysis buffer freezes, wait until it thaws before proceeding to the next step.
    5. Keep sample on ice and homogenize with 10-20 strokes of the pestle.
    6. Transfer sample into a 1.5 ml microcentrifuge tube and determine the protein concentration.
      Note: The concentration should be at least 4 µg/µl.
    7. Divide into 50-100 μl aliquots, snap freeze in liquid nitrogen and store at -80 °C for later use.
    8. Prepare gradient gel (gel dimension: 18 x 16 x 0.15 cm) from 14 ml of each of the two separation solutions (4% and 14% acrylamide) with a gradient mixer. Overlay gel with stacking solution prior it gets solid. Let polymerize at room temperature, and then cool the gel down to 4 °C.
    9. To prepare for BN-PAGE, let the sample thaw on ice and then combine 10-25 µl sample (50-100 µg) of it with 25 µl sample buffer, 17 µl 2% β-DM solution and 4.5 µl BN-Loading buffer.
      Note: The Bradford assay was used to determine protein concentration.
    10. Incubate sample on ice for 10 min.
    11. Centrifuge sample for 10 min at 17,500 x g and 4 °C.
    12. Load supernatant into wells and run the gel overnight (16-18 h) at 50 V constant voltage at 4 °C (or after the dye front has migrated about one third).
    13. Replace blue cathode buffer to cathode buffer without dye.
    14. Continue the electrophoresis at 10 mA constant current (~600 V) until the dye runs off (4-5 h).
    15. Remove gel from glass plates and soak gel in precooled (10 °C) transfer buffer for approx. 30 min.
    16. Transfer proteins onto PVDF membrane using semi-dry blotting apparatus (for 3 h at 0.8-3 mA/cm2 at room temperature).
    17. Destain membrane in 100% methanol for 1-3 min and immediately rinse with water.
    18. Block membrane overnight with 5% casein in 1x TBST buffer at 4 °C.
    19. Rinse membrane briefly with water and then proceed with immunoprobing according to a standard protocol (Sambrook and Russell, 2001).

  2. Sucrose gradient sedimentation analysis. The protocol for sample preparation described above can be used with the following modifications:
    1. Harvest tissue from 2-3 seedlings (second whole leaves).
    2. Use 1.5 ml buffer for lysis (step A3).
    3. After homogenization (step A5) on ice transfer sample into a 15 ml conical tube.
    4. Sonicate sample (tip width 2-3 mm) for three cycles of 10 sec at 20% power output. Let samples sit on ice for 1-2 min between sonication (Note 1).
    5. Transfer lysate into a 2.0 ml microcentrifuge tube and centrifuge at 20,000 x g for 30 min at 4 °C to remove insoluble material. Take an aliquot of each fraction and detect individual PEP subunits by immunoblot analysis (Note 1).
    6. Transfer supernatant into a fresh 15 ml tube and determine protein concentration.
      Note: Concentration should be at least 3 µg/µl.
    7. Snap freeze sample (supernatant) in liquid nitrogen and store at -80 °C for later use.
    8. Thaw samples on ice for > 30 min and add 1.5 ml diluent solution. Diluent solution is used to dilute the glycerol concentration in the sample prior to sucrose gradient centrifugation.
    9. Concentrate sample to 1.0 ml using cut-off centrifugal filter device, when necessary.
    10. Prepare 12-ml continuous gradients of 10 to 30% sucrose (from 6 ml each) in 14 ml polyallomer centrifugal tubes with a gradient mixer (sucrose gradient preparation in Zhu, 2012).
    11. Load slowly 1.0 ml sample (approx. 5 mg) on top of a gradient and centrifuge tubes for 15 h at 146,000 x g in an SW-40 rotor (4 °C).
    12. Carefully collect 500-µl-fractions from the top of the gradient.
    13. Use equal volumes (30-50 µl) of every second fraction for SDS-PAGE and subsequent immunoblot analysis with an antibody specific for a PEP subunit.


      Figure 1. Flowchart of experimental procedure

Representative data


Figure 2. Separation of protein complexes by BN-PAGE and subsequent analyses of PEP complex assembly by immunoblotting with antibodies directed against the subunits RpoA and pTAC12. Approximately 50 µg of total leaf protein from the base section of second leaves were loaded per lane. RpoA, RNA polymerase alpha subunit; pTAC12, plastid Transcriptionally Active Chromosome 12 (Note 2).


Figure 3. Sucrose-gradient sedimentation. Total leaf proteins from basal half of second leaf were solubilized in BN-Lysis buffer with sonication and soluble fractions subjected to sucrose gradient centrifugation for analysis of PEP-complex assembly (Pfalz et al., 2014). Fractions were collected and immunoblotted with antibodies against pTAC12, RpoA and Rubisco (RbcL). Respective fractions containing Rubisco, PEP (plastid-encoded RNA polymerase), and TAC (transcriptionally active chromosome) (Note 2) are indicated by bars.

Notes

  1. The DNA-bound PEP fraction (TAC) is tightly associated with the thylakoid membranes. It can be released by mild sonication. To validate efficient release of PEP from membranes by sonication take an aliquot of each fraction and detect individual PEP subunits by immunoblot analysis.
  2. The TAC complex does not enter the separation gel on conventional BN-Gels, and therefore cannot be immunodetected. The detected proteins by BN-PAGE represent the soluble PEP complex. It is expected to see a shift towards small size fractions if the complex is partially assembled. In case that the proteins are not properly assembled into the full PEP complex the appropriate bands will disappear (PEP: ~1,000 kDa).

Recipes

Note: All buffers should be stored at 4 °C prior use unless otherwise noted.

  1. BN-Lysis buffer
    100 mM Tris-HCl (pH 7.3)
    10 mM MgCl2
    25% (v/v) glycerol
    1% (v/v) Triton X-100
    5 mM β-mercaptoethanol
    1x Protease inhibitor cocktail
  2. 3x BN-Gel buffer
    150 mM Bis-Tris (pH 7.0)
    1.5 M ε-aminocaproic acid
  3. 4.5% Separating gel (prepare fresh)
    1.80 ml Acrylamide/Bisacrylamide (29:1)
    5.40 ml 3x BN-Gel Buffer
    1.80 ml 75% Glycerol
    7.13 ml dH2O
    35 μl 10% APS
    35 μl TEMED
  4. 14% Separating gel (prepare fresh)
    5.60 ml Acrylamide/Bisacrylamide (29:1)
    5.40 ml 3x BN-Gel Buffer
    4.6 ml 75% glycerol
    330 µl dH2O
    35 μl 10% APS
    35 μl TEMED
  5. 4% Stacking gel (prepare fresh)
    2.1 ml Acrylamide/Bisacrylamide (29:1)
    7.00 ml 3x BN-Gel Buffer
    75% glycerol
    11.79 ml dH2O
    50 µl 10% APS
    60 µl TEMED
  6. Cathode buffer blue
    0.5 M tricine
    150 mM Bis-Tris (pH 7.0)
    0.02% Coomassie blue G250
  7. Cathode buffer
    0.5 M tricine
    150 mM Bis-Tris (pH 7.0)
  8. Anode buffer
    0.5 M Bis-Tris (pH 7.0)
  9. 2x BisTris ACA (can be stored at -20 °C)
    200 mM Bis-Tris (pH 7.0)
    1 M ε-aminocaproic acid
  10. Sample buffer (can be stored at -20 °C)
    125 µl 100 mM Bis-Tris (pH 7.0)
    250 µl 40% glycerol
    20 µl 25x Protease inhibitor cocktail
    5 µl 1M NaF
    100 µl dH2O
  11. 2% n-dodecyl-β-D-maltopyranoside (β-DM) solution (can be stored at -20 °C)
    125 µl 100 mM Bis-Tris (pH 7.0)
    500 µl 40% glycerol
    200 µl 10% β-DM (in dH2O)
    50 µl 25x Protease inhibitor cocktail
    125 µl dH2O
  12. BN-Loading buffer (can be stored at -20 °C)
    50 mg Coomassie blue G250
    500 µl 2x BisTris ACA
    400 µl 75% saccharose
    50 µl dH2O
  13. Transfer buffer
    0.5% SDS
    25 mM Tris-HCl (pH 7.6)
    192 mM glycine
    10% methanol
  14. 1x TBST
    25 mM Tris-HCl (pH 7.5)
    125 mM NaCl
    0.10% Tween-20
  15. Low sucrose solution (should be made fresh each time and stored at 4 °C prior to use)
    10% (w/v) sucrose
    30 mM HEPES-KOH (pH 8.0)
    100 mM KOAc
    10 mM Mg(OAc)2
    5 mM dithiothreitol
  16. High sucrose solution (should be made fresh each time and stored at 4 °C prior to use)
    30% (w/v) sucrose
    30 mM HEPES-KOH (pH 8.0)
    100 mM KOAc
    10 mM Mg(OAc)2
    5 mM dithiothreitol
  17. Diluent solution (can be stored at -20 °C)
    30 mM HEPES-KOH (pH8.0)
    200 mM KOAc
    20 mM Mg(OAc)2
    2 mM dithiothreitol
    1x Protease inhibitor cocktail

Acknowledgments

The work was supported by the DFG (PF667-4). This work is adapted from the work previously published (Pfalz et al., 2015). BN-PAGE is performed essentially as described by Schägger and von Jagow.

References

  1. Liere, K., Weihe, A. and Borner, T. (2011). The transcription machineries of plant mitochondria and chloroplasts: Composition, function, and regulation. J Plant Physiol 168(12): 1345-1360.
  2. Pfalz, J. and Pfannschmidt, T. (2013). Essential nucleoid proteins in early chloroplast development. Trends Plant Sci. 18(4): 186-194.
  3. Pfalz, J., Holtzegel, U., Barkan, A., Weisheit, W., Mittag, M. and Pfannschmidt, T. (2015). ZmpTAC12 binds single-stranded nucleic acids and is essential for accumulation of the plastid-encoded polymerase complex in maize. New Phytol 206(3): 1024-1037.
  4. Sambrook, J. and Russell, D. W. (2001). Molecular cloning: A laboratory manual. 3rd edition. Cold Spring Harbor.
  5. Schagger, H. and von Jagow, G. (1991). Blue native electrophoresis for isolation of membrane protein complexes in enzymatically active form. Anal Biochem 199(2): 223-231.
  6. Zhelyazkova, P., Sharma, C. M., Forstner, K. U., Liere, K., Vogel, J. and Borner, T. (2012). The primary transcriptome of barley chloroplasts: numerous noncoding RNAs and the dominating role of the plastid-encoded RNA polymerase. Plant Cell 24(1): 123-136.
  7. Zhu, H. (2012). Sucrose gradient analysis. Bio-protocol Bio101: e194.

简介

质体编码聚合酶(PEP)代表成熟叶绿体中的主要转录机制(Liere等人,2011; Zhelyazkova等人,2012)。 这种多亚基复合物的正确装配对于植物生长和发育是重要的(Pfalz和Pfannschmidt,2013)。 PEP聚合酶可以从可溶性和膜结合(也称为转录活性染色体,TAC)级分中纯化。 蓝色使用天然聚丙烯酰胺凝胶电泳(BN-PAGE)和蔗糖梯度沉淀,随后进行免疫印迹分析来检测PEP复合物组装体的状态。

关键字:Polymerase丰富的质体编码(PEP), RNA聚合酶装配, 蓝色变性PAGE(BN-PAGE), 蔗糖密度梯度离心法, 质体蛋白复合物

材料和试剂

  1. 材料
    1. 一次性无RNase移液器吸头(1-200μl和100-1,000μl通用吸头)
    2. 无RNase的微量离心管(1.5ml和2.0ml通用试管)
    3. 15 ml锥形管
    4. Amicon Ultra-15离心过滤器单元,10KDa(Merck Millipore Corporation,目录号:UFC901024)
    5. PVDF膜(Carl Roth,Roti-氟,目录号:2803.1)
    6. 聚丙烯离心管,14×95mm(Beckman,目录号:331374)

  2. 植物材料
    1. 玉米幼苗在26-28℃下在土壤中生长7-10天,周期为16小时光/8小时黑暗

  3. 抗体
    1. 抗ZmpTAC12(定制抗体)(Biogenes)
    2. 抗ZmRpoA(定制抗体)(Biogenes)
    3. 山羊抗兔IgG HRP-连接(Sigma-Aldrich,目录号:A6154)
  4. 试剂
    1. 液氮
    2. 丙烯酰胺,Gel 40(29:1)(Carl Roth,Rotiphorese ,目录号:A515.1)
    3. 将过硫酸铵(APS)(NH 4)2 SubS 2 O 8(Sigma-Aldrich,目录号:248614-500G-D)
    4. 双 - (2-羟乙基) - 亚氨基 - 三 - (羟甲基) - 甲烷,Bis-Tris(Carl Roth,目录号:9140.3)
    5. β-巯基乙醇(Carl Roth,目录号:4227.1)
    6. Bradford,Roti Nanoquant(Carl Roth,目录号:K880.1)
    7. 考马斯亮蓝G250(Sigma-Aldrich,目录号:B0770-5G)
    8. 1,4-二硫苏糖醇,DTT(Carl Roth,目录号:6908.1)
    9. ε-氨基己酸(Carl Roth,目录号:3113.2)
    10. 甘油(Carl Roth,目录号:6962.3)
    11. 甘氨酸(Carl Roth,目录号:3187.4)
    12. HEPES(Carl Roth,目录号:9105.3)
    13. 醋酸镁四水合物,Mg(CH 3 COO)2·4H 2 O(Carl Roth,目录号:0275.2)
    14. 氯化镁,MgCl 2(Carl Roth,目录号:KK36.3)
    15. 甲醇(Carl Roth,目录号:P717.1)
    16. 正十二烷基β-D-麦芽糖苷,β-DM(Sigma-Aldrich,目录号:D4641-1G)
    17. N,N,N',N' - 四甲基乙二胺(TEMED)(Carl Roth,目录号:2367.1)
    18. N-Tris-(羟甲基) - 甲基 - 甘氨酸,Tricin(Carl Roth,目录号:6977.3)
    19. BlueEasy Prestained Protein Marker(Nippon Genetics,目录号:MWP06)
    20. 乙酸钾K(CH 3 COO)(Carl Roth,目录号:T874.1)
    21. 氢氧化钾(Carl Roth,目录号:7986.1)
    22. 蛋白酶抑制剂混合物(cOmplete)
      注意:目前,它是"Sigma-Aldrich,cOmplete TM ,目录号: 000000011836153001"。
    23. 三 - (羟甲基) - 氨基甲烷,TRIS(Carl Roth,目录号:4855.3)
    24. Triton X-100(Carl Roth,目录号:3051.2)
    25. Tween-20(Carl Roth,目录号:9127.1)
    26. 氯化钠,NaCl(Carl Roth,目录号:9265.2)
    27. 十二烷基硫酸钠,SDS(Carl Roth,目录号:4360.2)
    28. 氟化钠,NaF(Carl Roth,目录号:4503.2)
    29. D-蔗糖[D(+) - 蔗糖] Carl Roth,目录号:4621.2)
  5. 缓冲区
    1. BN-Lysis缓冲液(见配方)
    2. 3x BN-Gel缓冲液(见配方)
    3. 4.5%分离凝胶(见配方)
    4. 14%分离凝胶(见配方)
    5. 4%堆叠凝胶(见配方)
    6. 阴极缓冲液蓝色(见配方)
    7. 阴极缓冲液(见配方)
    8. 阳极缓冲液(见配方)
    9. 2x BisTris ACA(ACA,氨基己酸)(参见配方)
    10. 示例缓冲区(参见配方)
    11. 2%正十二烷基-β-D-吡喃麦芽糖苷(β-DM)溶液(参见配方)
    12. BN加载缓冲区(参见配方)
    13. 传输缓冲区(请参阅配方)
    14. 1x TBST缓冲区(参见配方)
    15. 低蔗糖溶液(见配方)
    16. 高蔗糖溶液(见配方)
    17. 稀释溶液(参见配方)

设备

  1. 砂浆和杵
  2. 超声波仪(BANDELIN electronic GmbH& Co.,型号:Sonopuls HD 2200)
  3. 微量离心机(Beckman,型号:Avanti JXN-30)
  4. 超速离心机(Sorvall Discovery 90SE)
    注意:此产品已由制造商停产。
  5. SW40转子(Beckman)
  6. 梯度混合器(BIO-RAD,型号:385)
    注意:此产品已由制造商停产。
  7. 生物光度计(Eppendorf)
  8. 电泳装置(Hoefer,型号:SE600)
  9. Semi-Dry-Blotter(VWR International,PEQLAB Biotechnologie GmbH)

程序

  1. 蓝色天然聚丙烯酰胺凝胶电泳(BN-PAGE)分析
    1. 收集植物组织(一秒一叶的基底一半)到1.5毫升微量离心管,并迅速在液氮中冷冻。冷冻的组织可以在-80℃下储存1-2个月。
      注意:由于PEP亚基的表达在年轻时比成熟叶绿体高,因此在他的研究中使用来自幼叶部分(第二叶的基底一半)的蛋白质。
    2. 在预冷的研钵中用杵将组织研磨成在液氮中的细粉末。
    3. 让所有的液氮蒸发,然后立即加入200-400μl冰冷的BN-裂解缓冲液直接在研钵中的组织。在添加BN-Lysis缓冲液之前,不要让组织解冻。
    4. 如果裂解缓冲液冻结,请等待其解冻,然后再进行下一步骤
    5. 将样品保持在冰上,并用杵的10-20次冲击匀浆。
    6. 将样品转移到1.5毫升微量离心管,并确定蛋白质浓度 注意:浓度应至少为4μg/μl。
    7. 分成50-100μl等分试样,在液氮中快速冷冻,储存于-80℃备用
    8. 使用梯度混合器从14ml的两种分离溶液(4%和14%丙烯酰胺)中的每一种制备梯度凝胶(凝胶尺寸:18×16×0.15cm)。在凝固之前用堆叠溶液覆盖凝胶。在室温下聚合,然后将凝胶冷却至4℃。
    9. 为了准备BN-PAGE,让样品在冰上解冻,然后将10-25μl样品(50-100μg)与25μl样品缓冲液,17μl2%β-DM溶液和4.5μlBN-加载缓冲液。
      注意:使用Bradford测定法测定蛋白质浓度。
    10. 在冰上孵育样品10分钟。
    11. 在17,500×g和4℃下离心样品10分钟。
    12. 将上清液置于孔中,并在4℃下(或在染料前沿迁移约三分之一后)在50V恒定电压下运行凝胶过夜(16-18小时)。
    13. 将蓝色阴极缓冲液更换为无染料的阴极缓冲液。
    14. 在10mA恒定电流(?600V)下继续电泳,直到染料耗尽(4-5小时)
    15. 从玻璃板上取下凝胶,并在预冷(10℃)转移缓冲液中浸泡约1分钟。 30分钟。
    16. 使用半干印迹装置(在室温下在0.8-3mA/cm 2下3小时)将蛋白转移到PVDF膜上。
    17. 脱去膜在100%甲醇1-3分钟,立即用水冲洗
    18. 在4℃下在1×TBST缓冲液中用5%酪蛋白阻断膜过夜
    19. 用水简单冲洗膜,然后根据标准方案进行免疫印迹(Sambrook和Russell,2001)。

  2. 蔗糖梯度沉降分析。上述样品制备方案可以使用以下修改:
    1. 收获组织从2-3苗(第二整叶)。
    2. 使用1.5 ml缓冲液进行裂解(步骤A3)。
    3. 在将冰转移样品均匀化(步骤A5)到15ml锥形管中之后
    4. 超声波样品(尖端宽度2-3毫米)三个周期为10秒,20%功率输出。让样品置于冰上超声1-2分钟(注1)。
    5. 将裂解物转移到2.0ml微量离心管中并在4℃下以20,000×g离心30分钟以除去不溶性物质。取每个级分的等分试样并通过免疫印迹分析检测单个PEP亚基(注1)
    6. 将上清液转移到新鲜的15ml管中并测定蛋白质浓度 注意:浓度应至少为3μg/μl。
    7. 将冷冻样品(上清液)置于液氮中,并储存在-80℃备用。
    8. 在冰上解冻样品> 30分钟并加入1.5ml稀释液。稀释溶液用于在蔗糖梯度离心之前稀释样品中的甘油浓度
    9. 如有必要,使用截止离心过滤装置将样品浓缩至1.0ml
    10. 准备12毫升连续梯度的10%至30%蔗糖(从每个6毫升)在14毫升多聚集离心管与梯度混合器(蔗糖梯度制备朱,2012)。
    11. 在SW-40转子(4℃)中缓慢加载1.0ml样品(约5mg)至梯度顶部并在146,000×g离心管中离心15小时。
    12. 从梯度顶部小心收集500μl级分。
    13. 使用等体积(30-50微升)每秒的馏分进行SDS-PAGE和随后的免疫印迹分析与特异于PEP亚单位的抗体。


      图1.实验程序流程图

代表数据


图2.通过BN-PAGE分离蛋白质复合物并通过用针对亚基RpoA和pTAC12的抗体进行免疫印迹分析PEP复合体装配物。从第二个基底部分获得约50μg的总叶蛋白叶每个泳道加载。 RpoA,RNA聚合酶α亚基; pTAC12,plastid Transcriptively Active Chromosome 12(注释2)。


图3.蔗糖梯度沉降。来自第二叶的基底半部的总叶蛋白溶解于具有超声处理的BN-裂解缓冲液中,并将可溶性级分进行蔗糖梯度离心以分析PEP-复合体装配(Pfalz 等。,2014)。收集级分并用针对pTAC12,RpoA和Rubisco(RbcL)的抗体进行免疫印迹。用条表示含有Rubisco,PEP(质体编码的RNA聚合酶)和TAC(转录活性染色体)(注2)的各个级分。

笔记

  1. DNA结合的PEP部分(TAC)与类囊体膜紧密相关。它可以通过温和的超声处理释放。为了验证通过超声处理从膜中有效释放PEP,取各级分的等分试样,并通过免疫印迹分析检测单个PEP亚基。
  2. TAC复合物在常规BN-凝胶上不进入分离凝胶,因此不能进行免疫检测。通过BN-PAGE检测的蛋白质代表可溶性PEP复合物。如果复合物被部分组装,则预期将看到朝小尺寸级分的转变。在蛋白质没有正确装配成完整的PEP复合物的情况下,适当的条带将消失(PEP:?1,000kDa)。

食谱

注意:除非另有说明,否则所有缓冲液都应在4°C保存。

  1. BN-裂解缓冲液
    100mM Tris-HCl(pH7.3) 10mM MgCl 2/
    25%(v/v)甘油 1%(v/v)Triton X-100 5mMβ-巯基乙醇 1x蛋白酶抑制剂混合物
  2. 3x BN-Gel缓冲液
    150mM Bis-Tris(pH7.0) 1.5 Mε-氨基己酸
  3. 4.5%分离凝胶(新鲜制备)
    1.80ml丙烯酰胺/双丙烯酰胺(29:1) 5.40 ml 3x BN-Gel Buffer
    1.80ml 75%甘油
    7.13ml dH 2 O·dm / 35微升10%APS
    35微升TEMED
  4. 14%分离凝胶(新鲜制备)
    5.60ml丙烯酰胺/双丙烯酰胺(29:1)
    5.40 ml 3x BN-Gel Buffer
    4.6ml 75%甘油 330μldH 2 O
    35微升10%APS
    35微升TEMED
  5. 4%堆叠胶(新鲜制备)
    2.1ml丙烯酰胺/双丙烯酰胺(29:1)
    7.00 ml 3x BN-Gel Buffer
    75%甘油 11.79ml dH 2 O·dm / 50μl10%APS
    60μlTEMED
  6. 阴极缓冲蓝
    0.5 M tricine
    150mM Bis-Tris(pH7.0) 0.02%考马斯蓝G250
  7. 阴极缓冲区
    0.5 M tricine
    150mM Bis-Tris(pH7.0)
  8. 阳极缓冲区
    0.5 M Bis-Tris(pH 7.0)
  9. 2x BisTris ACA(可储存于-20°C)
    200mM Bis-Tris(pH7.0) 1 Mε-氨基己酸
  10. 样品缓冲液(可储存于-20°C)
    125μl100mM Bis-Tris(pH 7.0)
    250μl40%甘油 20μl25x蛋白酶抑制剂混合物
    5μl1M NaF
    100微升dH 2 O 2/d
  11. 2%正十二烷基-β-D-吡喃麦芽糖苷(β-DM)溶液(可储存于-20℃) 125μl100mM Bis-Tris(pH 7.0)
    500μl40%甘油
    200μl10%β-DM(在dH 2 O中)
    50μl25x蛋白酶抑制剂混合物
    125微升dH 2 O
  12. BN-加载缓冲液(可储存于-20°C)
    50mg考马斯蓝G250
    500μl2x BisTris ACA
    400μl75%蔗糖
    50微升dH 2 O
  13. 传输缓冲区
    0.5%SDS
    25mM Tris-HCl(pH7.6) 192 mM甘氨酸 10%甲醇
  14. 1x TBST
    25mM Tris-HCl(pH7.5) 125 mM NaCl 0.10%Tween-20
  15. 低蔗糖溶液(应每次新鲜制备,并在使用前储存于4°C) 10%(w/v)蔗糖 30mM HEPES-KOH(pH8.0) 100 mM KOAc
    10mM Mg(OAc)2
    5 mM二硫苏糖醇
  16. 高蔗糖溶液(每次应新鲜,使用前储存于4°C) 30%(w/v)蔗糖 30mM HEPES-KOH(pH8.0) 100 mM KOAc
    10mM Mg(OAc)2
    5mM二硫苏糖醇
  17. 稀释溶液(可储存于-20°C)
    30mM HEPES-KOH(pH8.0) 200mM KOAc
    20mM Mg(OAc)2
    2mM二硫苏糖醇 1x蛋白酶抑制剂混合物

致谢

这项工作得到了DFG(PF667-4)的支持。这项工作改编自以前发表的工作(Pfalz等人,2015年)。基本上如Sch?gger和von Jagow所述进行BN-PAGE。

参考文献

  1. Liere,K.,Weihe,A.和Borner,T。(2011)。  植物线粒体和叶绿体的转录机制:组成,功能和调节。植物生理 168(12):1345-1360。
  2. Pfalz,J。和Pfannschmidt,T。(2013)。  Essential nucleoid proteins in early chloroplast development。 Trends Plant Sci 。 18(4):186-194
  3. Pfalz,J.,Holtzegel,U.,Barkan,A.,Weisheit,W.,Mittag,M.和Pfannschmidt,T。(2015)。  ZmpTAC12结合单链核酸,并且对于质体编码的聚合酶复合物在玉米中的积累是必需的。 New Phytol 206(3):1024-1037。
  4. Sambrook,J。和Russell,D.W。(2001)。分子克隆:实验室手册。第3版。冷泉港。
  5. Schagger,H。和von Jagow,G。(1991)。 蓝色原生电泳用于分离酶活性形式的膜蛋白复合物/a>。 Anal Biochem 199(2):223-231。
  6. Zhelyazkova,P.,Sharma,CM,Forstner,KU,Liere,K.,Vogel,J.和Borner,T。(2012)。  大麦叶绿体的主要转录组:许多非编码RNA和质体编码的RNA聚合酶的主导作用。植物细胞 24(1):123-136。
  7. Zhu,H。(2012)。  蔗糖梯度分析。生物协议 Bio101:e194。

  • English
  • 中文翻译
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: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
引用:Pfalz, J. (2016). Investigating the Assembly Status of the Plastid Encoded Polymerase Using BN-PAGE and Sucrose Gradient Centrifugation . Bio-protocol 6(14): e1873. DOI: 10.21769/BioProtoc.1873.
提问与回复

(提问前,请先登录)bio-protocol作为媒介平台,会将您的问题转发给作者,并将作者的回复发送至您的邮箱(在bio-protocol注册时所用的邮箱)。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片或者视频的形式来说明遇到的问题。由于本平台用Youtube储存、播放视频,作者需要google 账户来上传视频。

当遇到任务问题时,强烈推荐您提交相关数据(如截屏或视频)。由于Bio-protocol使用Youtube存储、播放视频,如需上传视频,您可能需要一个谷歌账号。