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

Jeannette Pfalz

doi:10.21769/BioProtoc.1873

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Peer-reviewed

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

JP Jeannette Pfalz email

发布: 2016年07月20日第6卷第14期 DOI: 10.21769/BioProtoc.1873 浏览次数: 9119

评审: Marisa RosaAnonymous reviewer(s)

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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

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 )
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
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 )
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, cOmplete^TM, 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 )
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

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

Procedure

English

中文翻译

文章信息

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如何引用

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.