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Oct 2017
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Separation of Thylakoid Protein Complexes with Two-dimensional Native-PAGE
采用二维非变性PAGE分离类囊体蛋白复合物   

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Abstract

The hierarchical composition and interactions of the labile thylakoid protein complexes can be assessed by sequential 2D-native gel-electrophoresis system. Mild non-ionic detergent digitonin is used to solubilize labile protein super-and megacomplexes, which are then separated with first-dimension blue native polyacrylamide gel electrophoresis (1D-BN-PAGE). The digitonin derived protein complexes are further solubilized with stronger detergent, β-DM, and subsequently separated on an orthogonal 2D-BN-PAGE to release smaller protein subcomplexes from the higher-order supercomplexes. Here we describe a detailed method for 2D-BN-PAGE analysis of thylakoid protein complexes from Arabidopsis thaliana.

Keywords: Native gel electrophoresis (非变性凝胶电泳), Thylakoid membrane (类囊体膜), Thylakoid protein complexes (类囊体蛋白复合物), 2D-BN-PAGE (2D-BN-PAGE ), Light harvesting complex (光捕获复合物), Photosystem (光合系统), Photosynthesis (光合作用)

Background

Photosynthetic light reactions take place in the thylakoid membrane, which in higher plants is composed of appressed grana thylakoids and non-appressed stroma thylakoids. The light reactions are catalyzed by multi-subunit protein complexes photosystem (PS) I and II, cytochrome b6f and ATPase. PSII together with its light harvesting antenna complex (LHCII) is most abundant in grana-thylakoids and therefore spatially segregated from stroma thylakoid -located PSI-LHCI complexes (Andersson and Anderson, 1980). The interphase between the grana and stroma thylakoid is enriched in both photosystems (Albertsson, 2001; Suorsa et al., 2015). Mediated by light dependent reversible phosphorylation of LHCII and PSII proteins, the photosystems together with LHCII assemble into larger super- and megacomplexes. PSII core dimer together with two strongly and two moderately bound LHCII-trimers form large C2S2M2 supercomplexes, PSI together with loosely bound LHCII form PSI-LHCII supercomplexes, and finally, PSII and PSI together with L-LHCII form large PSII-LHCII-PSI megacomplexes (Caffarri et al., 2009; Pesaresi et al., 2009; Rantala et al., 2017).

Non-ionic detergents are generally used for isolation of native protein complexes from biological membranes. Mild detergent digitonin maintains weak interactions between protein complexes, but due to its bulky structure, it selectively solubilizes only the non-appressed regions of the thylakoids. However, when supplemented with low ionic strength salt, aminocaproic acid (ACA), digitonin gets access to the partition gap between two grana appressions and solubilizes the entire thylakoid membrane allowing the analysis of the overall organization of labile thylakoid protein complexes (Rantala et al., 2017). Solubilized protein complexes are supplemented with anionic Coomassie G-250 (CBB) dye that binds to the hydrophobic domains of protein complexes providing them with negative charge, and allows their electrophoretic separation according to molecular mass. Since the negative surface charges repel each other, CBB also prevents random protein complex aggregation.

Blue native PAGE (BN-PAGE) enables membrane protein complex separation in their native and functionally active form (Schägger and von Jagow, 1991). Coupling the 1D-BN-PAGE with a second (2D)-BN-PAGE allows the analysis of the subcomplex composition of the digitonin derived large protein super- and megacomplexes: The 1D-BN-gel lane containing the separated protein complexes is treated with slightly stronger detergent, n-dodecyl-β-D-maltoside (β-DM), which more effectively interferes with the interactions between protein complexes, particularly destroying the interaction of LHCII with the two photosystems. The lane is then subjected to the 2D-BN-PAGE for the separation of the dissociated subcomplexes. The composition of the subcomplexes can be further analyzed by electroblotting the 2D-gel and detecting specific proteins with antibodies or by cutting the lanes and subjecting the subcomplexes to denaturing 3D-SDS-PAGE.

This protocol describes optimized thylakoid protein complex isolation method and the analysis of the subcomplex composition of large protein super- and megacomplexes of Arabidopsis thaliana by 2D-BN-BN-PAGE. The method can be used for the analysis of the organization of the photosynthetic protein complexes and for the analysis of subcomplex composition of higher-order protein super- and megacomplexes.

Materials and Reagents

  1. Consumables
    1. Eppendorf microcentrifuge tubes 1.5 ml (Eppendorf, catalog number: 0030121694 )
    2. Falcon, Conical Centrifuge Tubes 15 ml (Corning, catalog number: 352096 )
    3. Culture tubes 5 ml (Lab Depot, catalog number: TLDT8301 )
    4. Finntip Pipette Tips (Finntip Flex 10, 250, 1,000 and 5 ml)
    5. Whatman chromatographic paper (GE Healthcare, catalog number: 1001-931 )
    6. PVDF membrane (Merck, catalog number: IPVH304F0 )

  2. Plant material
    Isolated thylakoids from 5-week old Arabidopsis thaliana (isolate thylakoids in the presence of 10 mM NaF in all buffers as described in Järvi et al., 2011)

  3. Reagents
    1. 6-aminocaproic acid (Sigma-Aldrich, catalog number: A2504 )
    2. Bis-Tris (Sigma-Aldrich, catalog number: B4429 )
    3. Glycerol (Avantor Performance Materials, J.T. Baker, catalog number: 7044 )
    4. Pefabloc SC (4-(2-Aminoethyl) benzenesulfonyl fluoride hydrochloride) (Roche Diagnostics, catalog number: 11585916001 )
    5. Sodium Fluoride (NaF) (Avantor Performance Materials, J.T. Baker, catalog number: 3688 )
    6. EDTA disodium salt (Avantor Performance Materials, J.T. Baker, catalog number: 1073 )
    7. Digitonin (Merck, Calbiochem, catalog number: 300410 )
    8. n-dodecyl-β-D-maltoside (Sigma-Aldrich, catalog number: D4641 )
    9. Serva Coomassie Blue G (SERVA Electrophoresis, catalog number: 35050 )
    10. Sucrose (Sigma-Aldrich, catalog number: S0389 )
    11. Tricine (Sigma-Aldrich, catalog number: T0377 )
    12. TEMED (Tetramethylethylenediamine) (Bio-Rad Laboratories, catalog number: 1610801 )
    13. Liquid nitrogen
    14. Methanol (VWR)
    15. Bovine Serum Albumin (BSA) (Sigma-Aldrich, catalog number: A7030 )
    16. Acrylamide (AA) (Sigma-Aldrich, catalog number: A9099 )
    17. (N,N'-Methylene)-Bis-Acrylamide (Bis-AA) (Merck, Omnipur, catalog number: 2610 )
    18. APS (Ammonium persulfate) (Bio-Rad Laboratories, catalog number: 1610700 )
    19. Tris (Sigma-Aldrich, catalog number: T1503 )
    20. Glycine (Fisher Scientific, catalog number: 10070150 )
    21. SDS (VWR, catalog number: 442444H )

  4. Antibodies
    1. Lhcb1 (Agrisera, catalog number: AS01 004 )
    2. Phospho (P)-Lhcb1 (Agrisera, catalog number: AS13 2704 )
    3. Lhcb2 (Agrisera, catalog number: AS01 003 )
    4. Phospho (P)-Lhcb2 (Agrisera, catalog number: AS13 2705 )

  5. Stock solutions
    1. Acrylamide solution A: 48% (w/v), 1.5% (w/v) Bis-acrylamide in MQ-water, store at 4 °C
    2. Acrylamide solution B: 20% (w/v), 5% (w/v) Bis-acrylamide in MQ-water, store at 4 °C
    3. APS (Ammonium persulfate): 5% (w/v) solution in MQ-water, store at 4 °C
    4. Digitonin stock solution: 10% (w/v) in MQ-water, store at -20 °C
    5. β-dodecyl maltoside: 10% (w/v) in MQ-water, store at -20 °C
    6. Glycerol: 75% (w/v) solution in MQ-water, store at 4 °C
    7. Pefabloc: 10 mg/ml (w/v) in MQ-water, store at -20 °C
    8. Sucrose: 75% (w/v) in MQ-water, store at -20 °C

  6. Buffers
    1. 3x Gel Buffer, store at 4 °C (see Recipes)
    2. 25BTH20G resuspension buffer, prepare fresh (see Recipes)
    3. ACA buffer (see Recipes)
    4. Detergent buffer A (4% Digitonin), prepare fresh (see Recipes)
    5. Detergent buffer B (1% β-DM), prepare fresh (see Recipes)
    6. CBB buffer, -20 °C (see Recipes)
    7. Anode buffer for BN, store at 4 °C (see Recipes)
    8. Blue cathode buffer for BN, store at 4 °C (see Recipes)
    9. Clear cathode buffer for BN, store at 4 °C (see Recipes)
    10. Transfer buffer, store at 4 °C (see Recipes)
    11. BN-PA: 3.5-12.5% separation gel, 3% stacking gel (see Recipes)

Equipment

  1. Dual gel caster with 10 x 8 cm plates (Hoefer, catalog number: SE215 )
  2. Hoefer gradient maker SG5 (or any gradient maker containing two 5 ml chambers)
  3. 0.75 mm T-spacers (Hoefer, catalog number: SE2119T-2-.75 )
  4. 1 mm T-spacers (Hoefer, catalog number: SE2119T-2-1.0 )
  5. Sample gel comb, 0.75 mm, 10 wells (Hoefer, catalog number: SE211A-10-.75 )
  6. 2D comb (flat) with a reference well, 1.0 mm thick
  7. Mighty Small SE250 vertical electrophoresis system (Hoefer, catalog number: SE250 )
  8. Ismatec IPC-pump
  9. Power supply, PowerPac HV (Bio-Rad Laboratories, catalog number: 164-5056 )
  10. Centrifuge (Eppendorf, model: 5424R )
  11. FinnpipetteTM F2 Variable Volume Single-Channel Pipettes
  12. Cold room (4 °C)
  13. Freezer (-20 °C)
  14. Photo scanner (e.g., Perfection V300 Photo, Epson, model: V300 )
  15. Rocker-Shaker (Biosan, model: MR-12, catalog number: BS-010130-AAI )
  16. Semi-dry blotting system (Hoefer, catalog number: TE77X )

Procedure

  1. Casting the BN-PA gel (linear gradient 3.5%-12.5% separation gel; 3% stacking gel)
    1. Assemble gel caster (e.g., Hoefer) with 10 x 8 cm plates (Figure 1A) according to manufacturer’s instructions. Any vertical electrophoresis system with any compatible casting unit can be used.
    2. Prepare the light (3.5% AA) and heavy (12.5% AA) buffers from the acrylamide stock solution A (48% acrylamide, 1.5% Bis-acrylamide) (see Recipes). Keep the solutions on ice to prevent untimely polymerization. Cast the gel between a glass plate and a notched aluminum oxide plate (10 x 8 cm) with a gradient maker (Figure 1B). Use 0.75 mm T-spacer for the 1D-BN-PAGE and 1 mm T-spacer for 2D-BN-PAGE. Allow the gradient gel to polymerize for 1-2 h at RT.
    3. Cast the stacking gel (3% acrylamide) using the acrylamide stock solution B (20% acrylamide, 5% Bis-acrylamide). Use an appropriate sample gel comb (1D-BN-PA gel: standard comb with 10 wells, 2D-BN-PA gel: 2D-comb). Allow the stacking gel to polymerize for 30-60 min. Remove the sample gel comb under MQ-water. Store the gel at 4 °C.
      PAUSE POINT: The gel can be stored at 4 °C at least for few days. The gel must not dry. 


      Figure 1. Equipment for casting gradient gels. A. Hoefer dual gel caster with all the required equipment; B. Gradient maker, IPC-pump and gel assembled and ready for gel casting.

  2. Solubilization of thylakoid membranes (Perform the solubilization under dim light)
    1. Resuspend thylakoids equivalent to 3-7 µg chlorophyll in ACA buffer to a chlorophyll concentration of 1 mg/ml.
    2. Add an equal volume of detergent buffer A (4% digitonin/ACA) on the sample and solubilize at RT with continuous gentle mixing for 10 min, e.g., in a shaker. The final chlorophyll concentration is 0.5 mg/ml.
    3. Pellet the insolubilized material by centrifugation at 18,000 x g, at 4 °C, for 20 min, remove the supernatant to a new Eppendorf tube.
    4. Add 1/10 of CBB-buffer to the sample and carefully mix with a pipet. Avoid making air bubbles.

  3. Separation of the protein complexes with 1D-BN-PAGE
    1. Assemble the gel to the vertical electrophoresis system and pour ~70 ml of blue cathode buffer to the gel tank (upper chamber), and ~100 ml of anode buffer to the lower chamber (Figure 2).


      Figure 2. Example of the gradient gel assembly. The gel is assembled in the SE 250 Mini-Vertical electrophoresis unit.

    2. Load thylakoids equivalent to 2-5 µg of chlorophyll to the sample well.
    3. Start the electrophoretic run by gradually increasing the voltage as follows:
      1. 75 V 30 min
      2. 100 V 30 min
      3. 125 V 30 min
      4. 150 V 60 min
      5. 175 V 30 min
      Perform the electrophoresis at 4 °C
    4. As the blue running front has moved about one third (at ~100-125 V) of the desired running distance, remove the blue cathode buffer from the upper chamber and replace it with a clear cathode buffer.
    5. Stop the electrophoresis, when the protein complexes (B1-B9) (see Figure 6A) are separated and the gel is clear from the blue cathode.
    6. Scan the gel with a photo scanner (keep the gel between the glass and alumina plates while scanning), remove the glass plate and carefully cut out the lane containing the separated protein complexes using a spacer (see sketch in Figure 3). Do not use scalpel or any other sharp object for cutting to avoid scratching the alumina plate.
      PAUSE POINT: It is possible to store the BN-gel strip at -80 °C, but do not shock-freeze the strip with liquid nitrogen.
      Notes: 
      1. The amount of chlorophyll loaded on the gel depends on the subsequent analysis: for immunoblotting, load 2 µg (or less) of chlorophyll and for the 3D-SDS-PAGE, load 5 µg or more chlorophyll.
      2. It is possible to also excise individual bands (complexes B1-B9) from the BN-PA-gel, re-solubilize the bands in Eppendorf-tubes with 1% β-DM and subject them to 2D-BN-PAGE. 


      Figure 3. Preparation of 1D-BN-strip for 2D-BN-PAGE. (1) After finishing 1D-BN-PAGE, remove the glass plate and excise the lane containing the protein complexes (B1-B9) using e.g., the T-spacer. (2) Carefully place the gel strip to a 5 ml culture tube containing 2 ml of detergent buffer B (1% β-DM). Solubilize in a shaker for 40 min. (3) Place the gel strip on the second BN-PA gel (the large well) and assemble the gel to the electrophoresis tank. Perform the 2D-electrophoresis as described for 1D-BN-PAGE.

  4. 2D-BN-PAGE
    1. Place the gel strip in a culture tube (5 ml) and add 2 ml of detergent buffer B (1% β-DM).
    2. Solubilize the strip for 40 min at 4 °C in gentle rocking to allow even solubilization of the strip (e.g., 20 rpm).
    3. Place the strip on the second BN-PA gel (see sketch in Figure 3) and assemble the gel to the electrophoresis tank.
    4. Perform the electrophoresis as described for the 1D-BN-PAGE.
    5. Scan the gel after the electrophoresis whit a photo scanner (see sketch of the 2D-BN-PAGE in Figure 4, the flowchart demonstrating the follow-up paths in Figure 5 and scanned image of the representative gel in Figure 6B).


      Figure 4. 2D-BN-PAGE separation of thylakoid protein complexes. Protein complexes B1-B9 (green bands on the upper horizontal gel slice) were first separated according to their mass and shape on 1D-BN-PAGE. After separation the gel slice was subjected to 2D-BN-PAGE during which the complexes (B1-B9) are fractionated into subcomplexes (narrow green bands on the 2D-BN-gel). The complexes on the diagonal (dashed line) represent complexes that have preserved their mass, whereas the complexes below the diagonal are subcomplexes (of B1-B9) that have been disconnected during second solubilization and 2D-BN-PAGE.


      Figure 5. Flowchart of the experimental procedure

  5. Western blotting of 2D-native gels
    1. Place the 2D-BN-PA-gel to transfer buffer and incubate for 30 min.
    2. Activate the PVDF membrane with 100% methanol and place the membrane to transfer buffer. Soak six Whatman filter papers in the transfer buffer.
    3. Assemble three papers on the electrode (anode). Place the membrane on top of the papers and the gel on top of the membrane. Finally, place the three papers on top of the gel and mount the cathode on top. Set the current for 1 mA/cm2 gel area, and transfer for at least 1 h.
    4. Destain the membrane with 100% methanol.
    5. Block the membrane with 5% BSA for 1 h.
    6. Perform immunostaining with specific antibodies (see representative blots from Figure 6C).
      1. Lhcb1, Lhcb2, Lhcb3
      2. P-Lhcb1 and P-Lhcb2

  6. 3D-SDS-PAGE
    1. Cut the lanes containing the subcomplexes (corresponding to complexes B1-B9).
    2. Protein solubilization and separation on SDS-PAGE are as described for the 2D-SDS-PAGE analysis in Järvi et al., 2011.
    3. After electrophoresis, visualize the protein with Sypro Ruby Stain or with silver staining according to Blum et al., 1987 (see representative 3D gels from Figure 6D, the nine gels correspond to complexes B1-B9).

Data analysis

  1. For all the experiments (2D-BN-PAGE, 3D-SDS-PAGE, Western blotting) we use three biological replicates.
  2. No image processing was performed. The identification of the protein complexes (on native gels) and individual proteins (on 3D-SDS gels) is based on mass spectrometry analysis according to (Aro et al., 2005 ; Suorsa et al., 2015).
  3. The representative data (Figure 6) is from the original research article (Rantala et al., 2017).


    Figure 6. The representative results from the analysis of the subcomplex and subunit composition of thylakoid protein complexes. A. Protein complexes (B1-B9) of digitonin solubilized thylakoids after 1D-BN-PAGE separation. B. 2D-BN-PAGE separation of the subcomplexes obtained by re-solubilizing the 1D-strip with β-DM. Ctrl represents thylakoids directly solubilized with 1% DM. C. The Lhcb-protein localization in protein subcomplexes was analyzed by electroblotting the 2D-BN-gels and immunodecorating the blots with Lhcb1, Lhcb2, Lhcb3 protein specific antibodies and with Phospho-Lhcb1-2 protein specific antibodies. D. The protein composition of the subcomplexes (derived from complexes B1-B9) was analyzed by cutting the lanes from 2D-BN-gel and subjecting the gel strips to 3D-BN-PAGE. The proteins are visualized with Sypro Ruby stain. This figure has been originally published in Rantala et al. (2017).

Notes

  1. The protocol is highly reproducible in our hands.
  2. Digitonin must be purified according to the manufacturer’s instructions. We had no problems with digitonin, but the detergent stock may precipitate easily when melted.
  3. The thylakoid isolation (protocol in Järvi et al., 2011) must be done from fresh leaves to obtain high-quality thylakoid protein complexes.
  4. Freezing and thawing the thylakoid sample several times will affect the stability of the protein complexes.

Recipes

Note: All the solutions are prepared in MQ water.

  1. 3x Gel buffer
    1.5 M 6-Aminocaproic acid
    150 mM Bis-Tris
  2. 25BTH20G
    25 mM Bis-Tris/HCl (pH 7.0)
    20% (w/v) glycerol
    0.25 mg/ml Pefabloc (add freshly)
    10 mM NaF (add freshly)
  3. ACA buffer
    25 mM Bis-Tris/HCl (pH 7.0)
    375 mM Aminocaproic acid
    1 mM EDTA
    0.25 mg/ml Pefabloc (add freshly from the stock solution)
    10 mM NaF (add freshly)
  4. Detergent buffer A
    4 % digitonin (w/v)
    25 mM Bis-Tris/HCl (pH 7.0)
    375 mM Aminocaproic acid
    1 mM EDTA
    0.25 mg/ml Pefabloc (add freshly from the stock solution)
    10 mM NaF (add freshly)
  5. Detergent buffer B
    1% β-dodecyl maltoside (w/v)
    25 mM Bis-Tris/HCl (pH 7.0)
    20% (w/v) glycerol and
    0.25 mg/ml Pefabloc (add freshly from the stock solution)
    10 mM NaF (add freshly)
  6. CBB buffer
    100 mM Bis-Tris/HCl (pH 7.0)
    0.5 M ACA
    30% (w/v) sucrose
    50 mg/ml Serva Blue G
  7. Anode buffer
    50 mM Bis-Tris/HCl (pH 7.0)
  8. Cathode buffer
    50 mM Tricine
    15 mM Bis-Tris/HCl (pH 7.0)
    0.01% Serva Blue G
  9. Transfer buffer
    39 mM glysine
    48 mM Tris
    0.0375% SDS
    20% methanol
  10. BN-PA: 3.5-12.5% separation gel, 3% stacking gel

    Notes:
    1. 10% aqueous APS must be made up fresh or stored frozen.
    2. The recipe is suitable for 0.75 mm gel, multiply the recipe by 1.5 for 1 mm gel.
    3. Prepare the solutions on ice to prevent untimely polymerization.

Acknowledgments

This research was financially supported by the Academy of Finland (project numbers 307335 and 303757), EU-funded Innovative Training Network (ITN) Solar Energy into Biomass (SE2B) Marie Skłodowska-Curie grant agreement (675006) and DPMLS. The protocol was adapted from publication (Rantala et al., 2017). We declare no conflicting or competing interests.

References

  1. Albertsson, P. (2001). A quantitative model of the domain structure of the photosynthetic membrane. Trends Plant Sci 6: 349-358.
  2. Andersson, B. and Anderson, J. M. (1980). Lateral heterogeneity in the distribution of chlorophyll-protein complexes of the thylakoid membranes of spinach chloroplasts. Biochim Biophys Acta 593: 427-440.
  3. Aro, E. M., Suorsa, M., Rokka, A., Allahverdiyeva, Y., Paakkarinen, V., Saleem, A., Battchikova, N. and Rintamäki, E. (2005). Dynamics of photosystem II: a proteomic approach to thylakoid protein complexes. J Exp Bot 56(411): 347-356.
  4. Blum, H., Beier, H. and Gross, H. J. (1987). Improved silver staining of plant proteins, RNA and DNA in polyacrylamide gels. Electrophoresis 8: 93-99.
  5. Caffarri, S., Kouřil, R., Kereïche, S., Boekema, E. J. and Croce, R. (2009). Functional architecture of higher plant photosystem II supercomplexes. EMBO J 28(19): 3052-3063.
  6. Järvi, S., Suorsa, M., Paakkarinen, V. and Aro, E. M. (2011). Optimized native gel systems for separation of thylakoid protein complexes: novel super- and mega-complexes. Biochem J 439: 207-214.
  7. Pesaresi, P., Hertle, A., Pribil, M., Kleine, T., Wagner, R., Strissel, H., Ihnatowicz, A., Bonardi, V., Scharfenberg, M., Schneider, A., Pfannschmidt, T. and Leister, D. (2009). Arabidopsis STN7 kinase provides a link between short- and long-term photosynthetic acclimation. Plant Cell 21(8): 2402-2423.
  8. Rantala, M., Tikkanen, M. and Aro, E. M. (2017). Proteomic characterization of hierarchical megacomplex formation in Arabidopsis thylakoid membrane. Plant J 92: 951-962.
  9. Schägger, H. and von Jagow, G. (1991). Blue native electrophoresis for isolation of membrane protein complexes in enzymatically active form. Anal Biochem 199: 223-231.
  10. Suorsa, M., Rantala, M., Mamedov, F., Lespinasse, M., Trotta, A., Grieco, M., Vuorio, E., Tikkanen, M., Jarvi, S. and Aro, E. M. (2015). Light acclimation involves dynamic re-organization of the pigment-protein megacomplexes in non-appressed thylakoid domains. Plant J 84: 360-373.

简介

不稳定的类囊体蛋白复合物的分级组成和相互作用可以通过连续的2D天然凝胶电泳系统来评估。 温和的非离子洗涤剂洋地黄皂苷用于溶解不稳定的蛋白质超级和巨型复合物,然后用第一维蓝色天然聚丙烯酰胺凝胶电泳(1D-BN-PAGE)分离。 将洋地黄皂苷衍生的蛋白质复合物用更强的去污剂β-DM进一步溶解,随后在正交的2D-BN-PAGE上分离,以从较高级的超复合物中释放较小的蛋白质亚复合物。 在这里,我们描述了来自拟南芥的类囊体蛋白复合物的2D-BN-PAGE分析的详细方法。

【背景】在类囊体膜中发生光合作用的光反应,在高等植物中,由贴壁的grana类囊体和非贴壁的基质类囊体组成。光反应由多亚基蛋白复合物光系统(PS)I和II,细胞色素b 6 f和ATP酶催化。 PSII及其光捕获天线复合物(LHCII)在grana-thylakoids中最为丰富,因此在空间上与基质类囊体定位的PSI-LHCI复合物隔离(Andersson和Anderson,1980)。格拉纳和基质类囊体之间的间期在两个光系统中都得到了丰富(Albertsson,2001; Suorsa et al。,2015)。通过光依赖性LHCII和PSII蛋白的可逆磷酸化介导,光系统与LHCII一起组装成更大的超级和超级复合物。 PSII核心二聚体与两个强烈和两个中等结合的LHCII-三聚体形成大的C2S2M2超复合物,PSI与松散结合的LHCII形成PSI-LHCII超复合物,最后,PSII和PSI与L-LHCII一起形成大的PSII-LHCII-PSI巨核复合物(Caffarri et al 。,2009; Pesaresi et al。,2009; Rantala et al。,2017)。

非离子型洗涤剂通常用于从生物膜中分离天然蛋白质复合物。温和的洗涤剂洋地黄皂苷维持蛋白质复合物之间的弱相互作用,但由于其庞大的结构,它仅选择性地溶解类囊体的非贴壁区域。然而,当补充低离子强度盐,氨基己酸(ACA)时,洋地黄皂苷可以进入两个纹状体之间的分隔间隙并溶解整个类囊体膜,从而可以分析不稳定类囊体蛋白复合物的整体组织(Rantala 等人。,2017)。溶解的蛋白质复合物补充有阴离子Coomassie G-250(CBB)染料,其结合蛋白质复合物的疏水结构域,为它们提供负电荷,并允许它们根据分子量进行电泳分离。由于负表面电荷相互排斥,CBB还防止随机蛋白质复合物聚集。

蓝色原位PAGE(BN-PAGE)能够以其天然和功能活性形式分离膜蛋白复合物(Schägger和von Jagow,1991)。将1D-BN-PAGE与第二(2D)-BN-PAGE偶联可以分析洋地黄皂苷衍生的大蛋白质超级和巨型复合物的亚复合物组成:含有分离的蛋白质复合物的1D-BN-凝胶泳道用稍强的去污剂, n - 十二烷基-β-D-麦芽糖苷(β-DM),它更有效地干扰蛋白质复合物之间的相互作用,特别是破坏LHCII与两种光系统的相互作用。然后对泳道进行2D-BN-PAGE以分离解离的亚复合物。可以通过电印迹2D凝胶并用抗体检测特定蛋白质或通过切割泳道并使亚复合物经历变性3D-SDS-PAGE来进一步分析亚复合物的组成。

该方案描述了优化的类囊体蛋白复合物分离方法和2D-BN-BN-PAGE分析拟南芥大蛋白超级和巨型复合物的亚复合物组成。该方法可用于分析光合蛋白复合物的组织,并用于分析高级蛋白质超级和超级复合物的亚复合物组成。

关键字:非变性凝胶电泳, 类囊体膜, 类囊体蛋白复合物, 2D-BN-PAGE , 光捕获复合物, 光合系统, 光合作用

材料和试剂

  1. 耗材
    1. Eppendorf微量离心管1.5 ml(Eppendorf,目录号:0030121694)
    2. Falcon,Conical Centrifuge Tubes 15 ml(Corning,目录号:352096)
    3. 培养管5 ml(Lab Depot,目录号:TLDT8301)
    4. Finntip移液器吸头(Finntip Flex 10,250,1,000和5 ml)
    5. Whatman色谱纸(GE Healthcare,目录号:1001-931)
    6. PVDF膜(默克,目录号:IPVH304F0)

  2. 植物材料
    来自5周龄拟南芥的分离的类囊体(在所有缓冲液中存在10mM NaF时分离类囊体,如Järvi等人,2011中所述)。
  3. 试剂
    1. 6-氨基己酸(Sigma-Aldrich,目录号:A2504)
    2. Bis-Tris(Sigma-Aldrich,目录号:B4429)
    3. 甘油(Avantor Performance Materials,J.T。Baker,目录号:7044)
    4. Pefabloc SC,(4-(2-氨基乙基)苯磺酰氟盐酸盐)(Roche Diagnostics,目录号:11585916001)
    5. 氟化钠(NaF)(Avantor Performance Materials,J.T。Baker,目录号:3688)
    6. EDTA二钠盐(Avantor Performance Materials,J.T.Baker,目录号:1073)
    7. Digitonin(默克,Calbiochem,目录号:300410)
    8. n -dodecyl-β-D-maltoside(Sigma-Aldrich,目录号:D4641)
    9. Serva Coomassie Blue G(SERVA电泳,目录号:35050)
    10. 蔗糖(Sigma-Aldrich,目录号:S0389)
    11. Tricine(Sigma-Aldrich,目录号:T0377)
    12. TEMED(四甲基乙二胺)(Bio-Rad Laboratories,目录号:1610801)
    13. 液氮
    14. 甲醇(VWR)
    15. 牛血清白蛋白(BSA)(Sigma-Aldrich,目录号:A7030)
    16. 丙烯酰胺(AA)(Sigma-Aldrich,目录号:A9099)
    17. (N,N'-亚甲基) - 双 - 丙烯酰胺(Bis-AA)(Merck,Omnipur,目录号:2610)
    18. APS(过硫酸铵)(Bio-Rad Laboratories,目录号:1610700)
    19. Tris(Sigma-Aldrich,目录号:T1503)
    20. 甘氨酸(Fisher Scientific,目录号:10070150)
    21. SDS(VWR,目录号:442444H)

  4. 库存解决方案
    1. 丙烯酰胺溶液A:MQ水中48%(w / v),1.5%(w / v)双丙烯酰胺,储存在4°C
    2. 丙烯酰胺溶液B:MQ水中20%(w / v),5%(w / v)双丙烯酰胺,储存在4°C
    3. APS(过硫酸铵):在MQ水中的5%(w / v)溶液,储存在4°C
    4. Digitonin原液:MQ水中10%(w / v),-20℃保存
    5. β-十二烷基麦芽糖苷:MQ水中10%(w / v),-20°C储存
    6. 甘油:在MQ水中的75%(w / v)溶液,储存在4°C
    7. Pefabloc:在MQ水中10 mg / ml(w / v),储存在-20°C
    8. 蔗糖:在MQ-水中的75%(w / v),储存在-20℃

  5. 缓冲区
    1. 3x凝胶缓冲液,储存在4°C(见食谱)
    2. 25BTH20G重悬浮缓冲液,准备新鲜(见食谱)
    3. ACA缓冲液(见食谱)
    4. 洗涤剂缓冲液A(4%Digitonin),准备新鲜(见食谱)
    5. 洗涤剂缓冲液B(1%β-DM),准备新鲜(见食谱)
    6. CBB缓冲液,-20°C(见食谱)
    7. BN的阳极缓冲液,储存在4°C(见食谱)
    8. 用于BN的蓝色阴极缓冲液,储存在4°C(见食谱)
    9. 用于BN的透明阴极缓冲液,储存在4°C(见食谱)
    10. 转移缓冲液,储存在4°C(见食谱)
    11. BN-PA:3.5-12.5%分离凝胶,3%堆积凝胶(见食谱)

  6. 抗体
    1. Lhcb1(Agrisera,目录号:AS01 004)
    2. Phospho(P)-Lhcb1(Agrisera,目录号:AS13 2704)
    3. Lhcb2(Agrisera,目录号:AS01 003)
    4. Phospho(P)-Lhcb2(Agrisera,目录号:AS13 2705)

设备

  1. 双凝胶脚轮,10 x 8 cm板(Hoefer,目录号:SE215)
  2. Hoefer梯度制造商SG5(或任何含有两个5毫升腔室的梯度制造商)
  3. 0.75 mm T型垫片(Hoefer,目录号:SE2119T-2-.75)
  4. 1 mm T型垫片(Hoefer,目录号:SE2119T-2-1.0)
  5. 样品凝胶梳,0.75 mm,10个孔(Hoefer,目录号:SE211A-10-.75)
  6. 2D梳子(扁平),带参考孔,1.0 mm厚
  7. Mighty Small SE250立式电泳系统(Hoefer,目录号:SE250)
  8. Ismatec IPC泵
  9. 电源,PowerPac HV(Bio-Rad Laboratories,目录号:164-5056)
  10. 离心机(Eppendorf,型号:5424R)
  11. Finnpipette TM F2可变容量单通道移液器
  12. 冷室(4°C)
  13. 冰箱(-20°C)
  14. 照片扫描仪(例如,Perfection V300照片,爱普生,型号:V300)
  15. Rocker-Shaker(Biosan,型号:MR-12,目录号:BS-010130-AAI)
  16. 半干印迹系统(Hoefer,目录号:TE77X)

程序

  1. 铸造BN-PA凝胶(线性梯度3.5%-12.5%分离凝胶; 3%堆积凝胶)
    1. 根据制造商的说明,用10 x 8 cm板(图1A)组装凝胶连铸机(例如,Hoefer)。可以使用任何具有任何兼容铸造单元的垂直电泳系统。
    2. 从丙烯酰胺储备溶液A(48%丙烯酰胺,1.5%双丙烯酰胺)中制备轻质(3.5%AA)和重质(12.5%AA)缓冲液(参见配方)。将溶液保存在冰上以防止不合时宜的聚合。用梯度制造器将凝胶浇铸在玻璃板和带缺口的氧化铝板(10×8cm)之间(图1B)。对于1D-BN-PAGE使用0.75 mm T-间隔物,对于2D-BN-PAGE使用1 mm T-间隔物。让梯度凝胶在室温下聚合1-2小时。
    3. 使用丙烯酰胺储备溶液B(20%丙烯酰胺,5%双丙烯酰胺)浇铸堆积凝胶(3%丙烯酰胺)。使用合适的样品凝胶梳(1D-BN-PA凝胶:标准梳子,10孔,2D-BN-PA凝胶:2D梳子)。使堆积凝胶聚合30-60分钟。在MQ-水下取下样品凝胶梳。将凝胶保存在4°C。
      PAUSE POINT:凝胶可以在4°C下储存至少几天。凝胶不能干燥。


      图1.用于浇铸梯度凝胶的设备。 A. Hoefer双凝胶连铸机,配备所有必需的设备; B.梯度制造商,IPC泵和凝胶组装并准备凝胶浇铸。

  2. 类囊体膜的溶解(在昏暗的光线下进行溶解)
    1. 在ACA缓冲液中重悬相当于3-7μg叶绿素的类囊体,叶绿素浓度为1 mg / ml。
    2. 在样品上加入等体积的洗涤剂缓冲液A(4%洋地黄皂苷/ ACA)并在室温下溶解,在振荡器中连续温和混合10分钟,例如。最终的叶绿素浓度为0.5毫克/毫升。
    3. 通过在18,000 x g ,4℃下离心20分钟沉淀不溶物质,将上清液移至新的Eppendorf管中。
    4. 将1/10的CBB缓冲液加入样品中,并用移液管小心地混合。避免制造气泡。

  3. 用1D-BN-PAGE分离蛋白质复合物
    1. 将凝胶装配到垂直电泳系统中,将~70 ml蓝色阴极缓冲液倒入凝胶罐(上室),将约100 ml阳极缓冲液倒入下室(图2)。


      图2.梯度凝胶组件示例凝胶在SE 250 Mini-Vertical电泳装置中组装。

    2. 将相当于2-5μg叶绿素的类囊体加载到样品孔中。
    3. 通过逐渐增加电压开始电泳运行,如下所示:
      1. 75 V 30分钟
      2. 100 V 30分钟
      3. 125 V 30分钟
      4. 150 V 60分钟
      5. 175 V 30分钟
      在4°C下进行电泳
    4. 由于蓝色运行前沿已移动所需运行距离的约三分之一(约100-125 V),从上部腔室移除蓝色阴极缓冲液并用透明阴极缓冲液替换。
    5. 当分离蛋白质复合物(B1-B9)(参见图6A)并且凝胶从蓝色阴极中清除时,停止电泳。
    6. 用扫描仪扫描凝胶(扫描时将凝胶保留在玻璃和氧化铝板之间),取下玻璃板,用垫片仔细切出含有分离的蛋白质复合物的泳道(见图3中的草图)。不要使用手术刀或任何其他尖锐物体进行切割,以免划伤氧化铝板。
      PAUSE POINT:可以在-80°C下储存BN-凝胶条,但不要用液氮冲洗条带。
      注意: 
      1. 凝胶上加载的叶绿素量取决于随后的分析:对于免疫印迹,加载2μg(或更少)的叶绿素,对于3D-SDS-PAGE,加载5μg或更多的叶绿素。
      2. 也可以从BN-PA-凝胶上切下单个条带(复合物B1-B9),用1%β-DM重新溶解Eppendorf管中的条带,并使它们进行2D-BN-PAGE。  


      图3.用于2D-BN-PAGE的1D-BN条带的制备(1)完成1D-BN-PAGE后,取出玻璃板并切除含有蛋白质复合物的泳道(B1- B9)使用例如,T-间隔物。 (2)小心地将凝胶条置于含有2ml洗涤剂缓冲液B(1%β-DM)的5ml培养管中。在振荡器中溶解40分钟。 (3)将凝胶条放在第二个BN-PA凝胶(大孔)上,并将凝胶装配到电泳槽中。按照1D-BN-PAGE所述进行2D电泳。

  4. 2D-BN-PAGE
    1. 将凝胶条放入培养管(5 ml)中,加入2 ml洗涤剂缓冲液B(1%β-DM)。
    2. 将条带在4°C温和摇动下溶解40分钟,使条带均匀溶解(例如,20 rpm)。
    3. 将条带放在第二个BN-PA凝胶上(见图3中的草图)并将凝胶装配到电泳槽中。
    4. 按照1D-BN-PAGE所述进行电泳。
    5. 电泳后用电子扫描仪扫描凝胶(参见图4中的2D-BN-PAGE草图,图5中的后续路径和图6B中代表性凝胶的扫描图像)。


      图4.类囊体蛋白复合物的2D-BN-PAGE分离。蛋白质复合物B1-B9(上部水平凝胶切片上的绿色条带)首先根据它们在1D-BN-的质量和形状分离页。分离后,将凝胶切片进行2D-BN-PAGE,在此期间将复合物(B1-B9)分级成亚复合物(2D-BN-凝胶上的窄绿色条带)。对角线上的复合物(虚线)表示保留其质量的复合物,而对角线下方的复合物是在第二次溶解和2D-BN-PAGE期间断开的亚复合物(B1-B9)。


      图5.实验程序流程图

  5. 2D原生凝胶的Western印迹
    1. 将2D-BN-PA-gel置于转移缓冲液中并孵育30分钟。
    2. 用100%甲醇活化PVDF膜并将膜置于转移缓冲液中。在转移缓冲液中浸泡六个Whatman滤纸。
    3. 在电极(阳极)上组装三张纸。将膜放在纸的顶部,将凝胶放在膜的顶部。最后,将三张纸放在凝胶顶部并将阴极安装在顶部。将电流设置为1 mA / cm 2 凝胶区域,并转移至少1小时。
    4. 用100%甲醇去除膜。
    5. 用5%BSA封闭膜1小时。
    6. 用特异性抗体进行免疫染色(参见图6C中的代表性印迹)。
      1. Lhcb1,Lhcb2,Lhcb3
      2. P-Lhcb1和P-Lhcb2

  6. 3D-SDS-PAGE
    1. 切割含有亚复合物的泳道(对应于复合物B1-B9)。
    2. SDS-PAGE上的蛋白质溶解和分离如Järvi et al。,2011中的2D-SDS-PAGE分析所述。
    3. 电泳后,根据Blum 等人,1987,用Sypro Ruby Stain或银染法观察蛋白质(参见图6D的代表性3D凝胶,9种凝胶对应于复合物B1-B9)。

数据分析

  1. 对于所有实验(2D-BN-PAGE,3D-SDS-PAGE,Western印迹),我们使用三个生物学重复。
  2. 没有执行图像处理。根据(Aro et al。,2005; Suorsa et al 。,2015)。
  3. 代表性数据(图6)来自最初的研究文章(Rantala et al。,2017)。


    图6.类囊体蛋白复合物的亚复合物和亚基组成分析的代表性结果。 A.在1D-BN-PAGE分离后,洋地黄皂苷溶解的类囊体的蛋白质复合物(B1-B9)。 B.通过用β-DM重新溶解1D-条带获得的亚复合物的2D-BN-PAGE分离。 Ctrl表示直接用1%DM溶解的类囊体。 C.通过电印迹2D-BN-凝胶并用Lhcb1,Lhcb2,Lhcb3蛋白特异性抗体和Phospho-Lhcb1-2蛋白特异性抗体对印迹进行免疫印迹来分析蛋白质亚复合物中的Lhcb蛋白定位。 D.通过从2D-BN-凝胶切割泳道并使凝胶条经受3D-BN-PAGE来分析亚复合物的蛋白质组成(衍生自复合物B1-B9)。用Sypro Ruby染色显示蛋白质。这个数字最初发表在Rantala et al。(2017)。

笔记

  1. 该协议在我们手中具有高度可重复性。
  2. 必须根据制造商的说明纯化Digitonin。我们对洋地黄皂苷没有任何问题,但洗涤剂原料在熔化时可能很容易沉淀。
  3. 类囊体分离(Järvi et al。,2011中的方案)必须从新鲜叶子中进行以获得高质量的类囊体蛋白质复合物。
  4. 将类囊体样品冷冻和解冻数次会影响蛋白质复合物的稳定性。

食谱

注意:所有溶液均在MQ水中制备。

  1. 3x凝胶缓冲液
    1.5 M 6-Aminocaproic acid
    150 mM Bis-Tris
  2. 25BTH20G
    25 mM Bis-Tris / HCl(pH 7.0)
    20%(w / v)甘油
    0.25 mg / ml Pefabloc(新鲜加入)
    10 mM NaF(新鲜添加)
  3. ACA缓冲区
    25 mM Bis-Tris / HCl(pH 7.0)
    375 mM氨基己酸
    1 mM EDTA
    0.25 mg / ml Pefabloc(新鲜加入原液)
    10 mM NaF(新鲜添加)
  4. 洗涤剂缓冲液A
    4%洋地黄皂苷(w / v)
    25 mM Bis-Tris / HCl(pH 7.0)
    375 mM氨基己酸
    1 mM EDTA
    0.25 mg / ml Pefabloc(新鲜加入原液)
    10 mM NaF(新鲜添加)
  5. 洗涤剂缓冲液B
    1%β-十二烷基麦芽糖苷(w / v)
    25 mM Bis-Tris / HCl(pH 7.0)
    20%(w / v)甘油和
    0.25 mg / ml Pefabloc(新鲜加入原液)
    10 mM NaF(新鲜添加)
  6. CBB缓冲区
    100 mM Bis-Tris / HCl(pH 7.0)
    0.5 M ACA
    30%(w / v)蔗糖
    50 mg / ml Serva Blue G
  7. 阳极缓冲区
    50 mM Bis-Tris / HCl(pH 7.0)
  8. 阴极缓冲剂
    50 mM Tricine
    15 mM Bis-Tris / HCl(pH 7.0)
    0.01%Serva Blue G
  9. 转移缓冲区
    39 mM glysine
    48 mM Tris
    0.0375%SDS
    20%甲醇
    10. BN-PA:3.5-12.5%分离凝胶,3%堆积凝胶*

    注意:
    1. 10%APS水溶液必须新鲜或冷冻保存。
    2. 该配方适用于0.75 mm凝胶,将配方乘以1.5,用于1 mm凝胶。
    3. 在冰上制备溶液以防止不合时宜的聚合。

致谢

该研究得到了芬兰科学院(项目编号307335和303757),欧盟资助的创新培训网络(ITN)太阳能生物质能(SE2B)MarieSkłodowska-Curie拨款协议(675006)和DPMLS的财政支持。该协议改编自出版物(Rantala et al。,2017)。我们声明没有相互冲突或相互竞争的利益。

参考

  1. Albertsson,P。(2001)。 光合膜结构域的定量模型。 趋势工厂Sci 6:349-358。
  2. Andersson,B。和Anderson,J.M。(1980)。 菠菜叶绿体类囊体膜叶绿素 - 蛋白质复合物分布的横向异质性。 Biochim Biophys Acta 593:427-440。
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  4. Blum,H.,Beier,H。和Gross,H.J。(1987)。 改善聚丙烯酰胺凝胶中植物蛋白,RNA和DNA的银染色。 Electrophoresis 8:93-99。
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  6. Järvi,S.,Suorsa,M.,Paakkarinen,V。和Aro,E。M.(2011)。 用于分离类囊体蛋白复合物的优化天然凝胶系统:新型超级和超级复合物。 Biochem J 439:207-214。
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  8. Rantala,M.,Tikkanen,M。和Aro,E。M.(2017)。 拟南芥类囊体膜中层状megacomplex形成的蛋白质组学表征。 Plant J 92:951-962。
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  10. Suorsa,M.,Rantala,M.,Mamedov,F.,Lespinasse,M.,Trotta,A.,Grieco,M.,Vuorio,E.,Tikkanen,M.,Jarvi,S。和Aro,EM(2015 )。 轻度驯化涉及在非贴壁类囊体结构域中动态重组色素 - 蛋白质巨核复合物。< / a> Plant J 84:360-373。
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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
引用:Rantala, M., Paakkarinen, V. and Aro, E. (2018). Separation of Thylakoid Protein Complexes with Two-dimensional Native-PAGE. Bio-protocol 8(13): e2899. DOI: 10.21769/BioProtoc.2899.
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