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May 2017

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Isolation of Thylakoid Membranes from the Cyanobacterium Synechocystis sp. PCC 6803 and Analysis of Their Photosynthetic Pigment-protein Complexes by Clear Native-PAGE
从蓝藻集胞藻sp. PCC 6803中分离类囊体膜并利用CN-PAGE非变性聚丙烯酰胺凝胶电泳分析光合色素蛋白复合物    

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Abstract

Cyanobacteria represent a frequently used model organism for the study of oxygenic photosynthesis. They belong to prokaryotic microorganisms but their photosynthetic apparatus is quite similar to that found in algal and plant chloroplasts. The key players in light reactions of photosynthesis are Photosystem I and Photosystem II complexes (PSI and PSII, resp.), large membrane complexes of proteins, pigments and other cofactors embedded in specialized photosynthetic membranes named thylakoids. For the study of these complexes a mild method for the isolation of the thylakoids, their subsequent solubilization and analysis is essential. The presented protocol describes such a method which utilizes breaking the cyanobacterial cells using glass beads in an optimized buffer. This is followed by their solubilization using dodecyl-maltoside and analysis using optimized clear-native gel electrophoresis which preserves the native oligomerization state of both complexes and allows the estimation of their content.

Keywords: Cyanobacteria (蓝藻), Photosynthesis (光合作用), Thylakoids (类囊体膜), Photosystem (光合系统), Clear-native gel (非变性聚丙烯酰胺凝胶电泳), Electrophoresis (电泳)

Background

Thylakoids are specialized membranes of algal and plant chloroplasts and cyanobacteria. The thylakoid lipid bilayer shares characteristic features with prokaryotic bacterial membranes but it is enriched in glycolipids, namely galactolipids, while the level of acidic lipids is relatively low and does not exceed 20% of the overall lipid content. Thylakoids contain a large number of proteins and their complexes and over 40% of them are involved in light-dependent reactions of photosynthesis. The next largest functional group includes proteins involved in protein targeting, processing and folding, oxidative stress response and translation. The most integral membrane proteins of thylakoids form large protein complexes which play an important role in the harvesting of light and its photochemical conversion. The key complexes performing this conversion are PSI and PSII. They both perform the light-driven primary charge separation resulting in the thylakoid-associated flow of electrons reducing NADP+ and generating a pH gradient used for the synthesis of ATP. Both complexes are subject to intensive research focused mainly on the elucidation of their function and underlying structural features as well as the mechanism of their assembly. For this research the isolation of native membranes and their detailed analysis as concerns the content, assembly and oligomerization state of both photosystems is essential. The existing and frequently used protocols for the isolation of cyanobacterial thylakoids either do not sufficiently preserve the native structure of fragile membrane complexes like Photosystem II dimers (e.g., Komenda and Barber, 1995), or the obtained preparations contain high concentrations of compounds (for instance 1 M glycine betaine) which are not compatible with the clear native gel electrophoresis (e.g., Gombos et al., 1994). The described protocol for thylakoid preparation is optimized from both points of view. The analysis of cyanobacterial membranes by clear native PAGE (CN PAGE) using a protocol modified from Wittig et al. (2007) gives direct information about the quantity and oligomeric (or assembly) state of multisubunit pigmented photosystem complexes with unprecedented speed and resolution. The isolation, solubilization, and analysis of membranes does not disrupt weak non-covalent interactions between pigments and proteins and therefore the gel with separated individual complexes can be directly scanned. Unlike denaturing SDS gels, no fixation, staining and destaining, or, blotting and immunodetection is needed for distinguishing between PSII and PSI (see Figure 2). Green PSI complexes do not show red fluorescence at room temperature and are present as dominant green bands in the gel while PSII complexes fluoresce and their green bands are hard to distinguish on the simple transparency scan. Thus, PSI quantification is done using the simple transparency scan of green non-fluorescent bands while PSII quantification is done using the red fluorescence scan of fluorescent bands.

Materials and Reagents

  1. Pipette tips
  2. 2 ml screw cap polypropylene micro vials (Biospec Products, catalog number: 3205)
  3. Hamilton® microsyringe series 700, 0.25 ml (Sigma, catalog number: 24538-U)
  4. Hamilton needle, 51 mm (Sigma, catalog number: 21746)
  5. Binder Clip (Deli 8584, Luma trading)
  6. Magnetic stirring bars with diameter 5 mm and length 20 mm
  7. Transparent plastic film
  8. 1.5 ml spectroscopic cuvettes (Fisherbrand PS, catalog number: 7755.0302)
  9. The glucose tolerant strain of the cyanobacterium Synechocystis sp. PCC6803 (GT-P, Tichý et al., 2016)
  10. Acrylamide for molecular biology (AA) (AppliChem Panreac, catalog number: A3812,1000)
  11. Bisacrylamide for molecular biology (BIS) (AppliChem Panreac, catalog number: A3636,0250)
  12. 6-aminohexanoic acid (ACA) (AppliChem Panreac, catalog number: A2266,500)
  13. Bis-Tris (AppliChem Panreac, catalog number: A1025.1000) 
  14. MES (AppliChem Panreac, catalog number: A0689.0250)
  15. Tricine (AppliChem Panreac, catalog number: A1085,1000)
  16. N,N,N',N'-tetramethylethylenediamine (TEMED) (Sigma, catalog number: T928-25ml)
  17. Ammonium persulfate (APS) (Sigma, catalog number: T3678-25g)
  18. Dithiothreitol (DTT) (AppliChem Panreac, catalog number: A1101,0025)
  19. SDS (Sigma, catalog number: 7172-100g)
  20. Glass beads with a diameter of 150-212 μm (Sigma, catalog number: G1145)
  21. n-Dodecyl-β-D-maltoside (DM) (AppliChem Panreac, catalog number: A0819,0005)
  22. Sodium deoxycholate (Sigma, catalog number: 30970-25g)
  23. Gel Filtration Markers Kit for Protein Molecular Weights (Sigma, catalog number: MWGF1000-1KT)
  24. CaCl2 (analytical grade)
  25. MgCl2 (analytical grade)
  26. Methanol (analytical grade)
  27. Sodium hydroxide (analytical grade)
  28. Hydrochloric acid (analytical grade)
  29. Glycerol (HPLC grade)
  30. Milli-Q de-ionized water (Merck Millipore, Ultrapure [Type 1] water)
  31. Buffer B (see Recipes)
  32. Gel buffer 6x (pH 7.6) (see Recipes)
  33. Acrylamide solution (AB) (see Recipes)
  34. Upper (Cathode) buffer 10x (see Recipes)
  35. Upper (Cathode) buffer 1x (see Recipes)
  36. Lower (Anode) buffer 10x (see Recipes)
  37. Lower (Anode) buffer 1x (see Recipes)
  38. Resolving gel and stacking gel (see Recipes)

Equipment

  1. Automatic pipettes (P1000, P200 and P10)
  2. Mini-beadbeater (Sigma, catalog number: Z249289)
  3. Magnetic stirrer Color Squid White (Thermo Fisher Scientific, catalog number: 6110.1005)
  4. GE gradient maker (Sigma, catalog number: GESG50) 
  5. Vortex (Velp Scientifica, model: RX3)
  6. Centrifuge (Hettich, model: Micro22 R)
  7. UV-VIS spectrophotometer (Shimadzu, model: UV3000)
  8. Cooled microcentrifuge (Eppendorf, model: 5415R) 
  9. PAGE apparatus (Bio-Rad, PROTEAN® II xi Cell)
  10. Power Supply (Bio-Rad, PowerPacTM HV High-Voltage)
  11. Cooled thermostat (Labio, model: CTB 06C)
  12. LAS 4000 camera (Fuji; for chlorophyll fluorescence scanning used LED blue light source 460 nm for excitation and R670 filter for emission)
  13. Scanner HP ScanJet G4050 (HP, model: G4050)
  14. pH meter (Sartorius, PB-11)
  15. FIM-150 flake ice maker (Biobase)

Software

  1. ImageJ bundled with Java 1.8.0_112 (https://imagej.nih.gov/ij/download.html)

Procedure

  1. The protocol requires 20 ml of Synechocystis PCC 6803 grown at 30 °C on an orbital shaker (about 60 rpm) under cool white fluorescent light of 35 μmol m-2 s-1 in liquid BG11 (Rippka et al., 1979) with an optical density of ~0.4-0.6 at 750 nm (see Notes). Collect the cyanobacterial cells by centrifugation at 2,300 x g for 10 min at ~4 °C, resuspend the cell pellet in 1.5 ml of Buffer B and transfer the resuspended cells into a 2 ml screw cap microtube. From this point keep the tubes on ice.
  2. Centrifuge the tube in the microcentrifuge at 9,300 x g for 2 min at 4 °C, discard the supernatant and resuspend the pellet in 0.15 ml of Buffer B and add the same volume of glass beads (measured separately in a graduated 0.5 ml microtube and added to the 2 ml microtube with the sample).
  3. Break the cells with three cycles of 20 s beating (max. speed) and 1 min cooling in a Mini-Beadbeater.
  4. Add 0.2 ml of Buffer B, vortex the tube for 2 s and immediately transfer the supernatant into a new pre-cooled 2 ml screw cap microtube, repeat five times.
  5. Centrifuge the tube at 400 x g for 1 min at 4 °C to remove unbroken cells and glass beads.
  6. Transfer the supernatant into a new pre-cooled 2 ml screw cap microvial and centrifuge at 16,000 x g for 20 min at 4 °C.
  7. Remove supernatant and resuspend the pellet in 0.2 ml of Buffer B using a stainless-steel piston from a 0.25 ml microsyringe (length 120 mm, diameter 2 mm): hold the tube with the pellet after removal of the supernatant in one and piston in the second hand, insert the piston into the tube down to the bottom and vortex for 15 s with inserted piston, then add 0.2 ml of the buffer and vortex again with piston inside for 10 s.
  8. Measure the chlorophyll (Chl) content in the suspension. Mix 13.5 µl of suspension with 1.5 µl of 10% DM, centrifuge at 16,000 x g for 5 min, take 10 µl of the supernatant and add to 990 µl of methanol in a screw cap microvial, vortex, centrifuge at 16,000 x g for 5 min, take the supernatant and measure absorbance at 720 and 666 nm, C (μg Chl/ml) = (A666 - A720) x 1,269 (Wellburn, 1994).
  9. Prepare the resolving gel (see Video 1).
    1. First assemble the gel cassettes from glass plates and 1 mm spacer according to the manufacturer’s instructions. 
    2. Then mix all components well (except for APS!) for each 4% and 14% resolving gel solution using magnetic stirrers and stirring bars (components of each solution are shown in Recipe 8, one column is one solution, the second and third column); then add APS to both solutions under constant mild stirring. 
    3. Pour the 4% solution together with stirring bar into the right compartment of the gradient maker located on the top of the magnetic stirrer. The gradient maker has an inlet attached to the silicone tube leading to the peristaltic pump and has a closed connecting channel to the second compartment. The outlet of the peristaltic pump is connected to the hypodermic needle attached to the upper part of the glass gel cassette using a binder clip. 
    4. Carefully open the connecting channel just enough to fill it with the 4% solution (if more solution is released, pipette it back to the right compartment) and close it again, and add now the 14% solution into the second (left) compartment of the gradient maker. 
    5. Remove 8.5 ml of the 14% solution with a pipette and add it directly to the bottom of the glass cassette for gel polymerization and immediately start pumping the remaining solution with a rate of about 10 ml per minute into the glass cassette and in parallel open the connecting channel between the compartments of the maker. Adjust mild stirring of the solution (2 turns per second). 
    6. After pouring the gradient, remove the needle, overlay the solution in the gel cassette with 0.5 ml of distilled water using a 0.25 ml syringe and wash the gradient maker and tubings with Milli-Q water. Watch the interface between the water and 4% solution until a clear border is formed and then remove the water with the syringe. 

    Video 1. Pouring the resolving gel

  10. Prepare the stacking gel. Add APS to the 10 ml 4% solution (right column in Recipe 8), pour to the top of the polymerized resolving gel, immerse the 10 well comb avoiding air bubbles and wait for polymerization; afterwards remove the comb and wash the wells with Milli-Q water.
  11. Attach the prepared gels into the cooling core, check if the connection is tight and not leaking with distilled water, pour out water and insert the core with gels into the electrophoresis chamber with 2 litres of 1x lower buffer, attach the silicon tubes of the cooling core to the thermostat, which is set to 4 °C and pour 200 ml of 1x upper buffer into the upper reservoir of the cooling core.
  12. For gel analysis take the samples containing 5 μg Chl, add a corresponding volume of Buffer B buffer to reach the same volume for all samples (usually 30 μl), add 1/10 of the volume of 10% DM and centrifuge immediately at 16,000 x g for 20 min at 4 °C.
  13. Take 25 μl of the solubilized membranes and load into the gel wells.
  14. For estimation of the molecular size of complexes, Gel Filtration Markers Kit for Protein Molecular Weights can be used, load 30 μl of the recommended mixture.
  15. Close the lid, attach it to the power supply and run in the constant current mode at 15 mA for 1.5-2 h with an upper voltage limit of 1,000 V (see Figure 1; during the run voltage gradually increases up to 1,000 V, after reaching this limit, the current goes down).


    Figure 1. The setup of the electrophoretic apparatus (lower right corner) with cooling thermostat (left) and power supply (upper right corner)

  16. After the pigmented front reaches a distance of about 8 cm from the bottom of the wells, switch off the power supply, remove the lid, detach the silicone cooling tubes and take out the cooling core with the gels from the electrophoretic chamber.
  17. Remove the gel from the glass cassette and wrap it into a transparent plastic film.
  18. Transfer it into the scanner to acquire the color image (300 dpi resolution, 48-bit color depth).
  19. Transfer the gel into the LAS4000 camera to take a 16 bit greyscale image in tiff format either in transparency mode to quantify green bands of PSI (Digitize DIA method, the lowest tray position, 3 s exposure, high resolution mode) or in epifluorescence mode using the blue LED excitation light (wavelength 460 nm) eliciting the chlorophyll (but not phycobiliprotein) fluorescence while the emitted light is passed through an R670 filter (long pass filter with the edge at 670 nm) to quantify PSII (Fluorescence method, the lowest tray position, 3 s exposure, high-resolution mode) (see Figure 2).


    Figure 2. Representative color scans (left pair) and chlorophyll fluorescence images (right pair) of the gel with separated Photosystem I and Photosystem II complexes from the cyanobacterium Synechocystis PCC 6803 GT-P (Tichý et al., 2016) and the Photosystem I-less mutant (Shen et al., 1993). Left two lanes show color scans, right ones Chl fluorescence scans with dominant bands of Photosystem II dimer and monomer. Blue bands (PBS frag.) represent fragments of phycobilisomes, the cyanobacterial antennae, fluorescence of which is not elicited by blue light.

  20. Quantify bands using ImageJ according to the user guide (https://imagej.nih.gov/ij/docs/guide/user-guide.pdf). The bands are first selected as regions of interest (ROIs) using the Area selection tools in Toolbar Tools, the background is subtracted using the processing tools and the intensity of the bands analyzed using the intensity statistics in the analysis tools.

Notes

Apart from the cyanobacterium Synechocystis PCC 6803 the described protocol has already been successfully tested for other cyanobacterial strains such as Synechococcus elongatus PCC 7942 and Synechococcus sp. PCC 7002. For the green algae Chlamydomonas reinhardtii, Chlorella sorokiniana and Scenedesmus quadricauda the protocol is identical, only larger glass beads with a diameter of 0.5 mm (Sigma) should be used for breaking the cells. The procedure for CN PAGE can also be used for isolated thylakoid membranes from plant chloroplasts.

Recipes

  1. Buffer B (50 ml stock)
    25 mM MES/NaOH buffer (pH 6.5)
    10 mM CaCl2
    10 mM MgCl2
    25% glycerol
    Divide into 10 ml aliquots and store frozen at -25 °C (stable for at least 6 months)
  2. Gel buffer 6x (pH 7.0, 100 ml)
    300 mM Bis-Tris/HCl buffer
    Adjust to pH 7.0 with 4 M HCl
    Store at 4 °C (stable for at least 6 months at 4 °C)
    Note: Usually 100 ml is prepared.
  3. Acrylamide solution (AB)
    50% AA
    1.33% BIS
    Milli-Q water
    Store at 4 °C (stable for at least 3 months)
    Note: Usually 500 ml is prepared.
  4. Upper (Cathode) buffer 10x
    0.5 M tricine
    150 mM Bis-Tris/HCl
    Adjust pH to 7.0 using 4 M HCl
    Store at 4 °C (stable for at least 6 months at 4 °C)
    Note: Usually 500 ml is prepared.
  5. Upper (Cathode) buffer 1x (200 ml for one PAGE)
    1. Add 20 ml of upper buffer 10x to 180 ml of Milli-Q water and 1 ml of 10% sodium deoxycholate to reach 0.05% final concentration
    2. Add 0.4 ml of 10% DM to reach 0.02% final concentration.
    Note: Prepare fresh before running.
  6. Lower (Anode) buffer 10x
    0.5 M Bis-Tris/HCl
    Adjust pH to 7.0 with 4 M HCl
    Store at 4 °C (stable for at least 6 months at 4 °C)
    Note: Usually 500 ml is prepared.
  7. Lower (Anode) buffer 1x (2 L)
    Dilute lower (Anode) buffer 10x to lower (Anode) buffer 1x with Milli-Q water
    Note: This solution is stable for at least 3 months at 4 °C and can be reused three times.
  8. Resolving gel and stacking gel
    Mix individual components (the first column from left) to get 4% (second column) and 14% (third column) solution for the preparation of the resolving gradient gel and 4% solution for the preparation of the stacking gel (fourth column) according to the following table:

Acknowledgments

This work was supported by the Czech Ministry of Education, Youth and Sports of the Czech Republic (project LO1416) and bilateral projects between the Academies of Sciences of the Czech Republic and Hungary (MTA-16-11).

Competing interests

We declare no conflict of interests.

References

  1. Gombos, Z., Wada, H. and Murata, N. (1994). The recovery of photosynthesis from low-temperature photoinhibition is accelerated by the unsaturation of membrane lipids: a mechanism of chilling tolerance. Proc Natl Acad Sci U S A 91(19): 8787-8791.
  2. Komenda, J. and Barber, J. (1995). Comparison of psbO and psbH deletion mutants of Synechocystis PCC 6803 indicates that degradation of D1 protein is regulated by the QB site and dependent on protein synthesis. Biochemistry 34(29): 9625-9631.
  3. Rippka, R., Deruelles, J., Waterbury, J. B., Herdman, M. and Stanier, R. Y. (1979). Generic assignments, strain histories and properties of pure cultures of cyanobacteria. Microbiology 111: 1-61.
  4. Shen, G., Boussiba, S. and Vermaas, W. F. (1993). Synechocystis sp PCC 6803 strains lacking photosystem I and phycobilisome function. Plant Cell 5(12): 1853-1863.
  5. Tichý, M., Beckova, M., Kopecna, J., Noda, J., Sobotka, R. and Komenda, J. (2016). Strain of Synechocystis PCC 6803 with aberrant assembly of photosystem II contains tandem duplication of a large chromosomal region. Front Plant Sci 7: 648.
  6. Wellburn, A. R. (1994). The spectral determination of chlorophylls a and b, as well as total carotenoids, using various solvents with spectrophotometers of different resolution. J Plant Physiol 144(3): 307-313.
  7. Wittig, I., Karas, M. and Schagger, H. (2007). High resolution clear native electrophoresis for in-gel functional assays and fluorescence studies of membrane protein complexes. Mol Cell Proteomics 6(7): 1215-1225.

简介

蓝藻代表了一种常用的模型生物,用于研究含氧光合作用。它们属于原核微生物,但它们的光合机构与藻类和植物叶绿体中的光合机构非常相似。光合作用光响应的主要参与者是光系统I和光系统II复合物(PSI和PSII,分别是),蛋白质,色素和其他辅因子的大型膜复合物,嵌入在称为类囊体的特化光合膜中。对于这些复合物的研究,一种温和的分离类囊体的方法,随后的溶解和分析是必不可少的。所提出的方案描述了这样一种方法,该方法利用在优化的缓冲液中使用玻璃珠破坏蓝细菌细胞。然后使用十二烷基 - 麦芽糖苷进行溶解并使用优化的透明 - 天然凝胶电泳进行分析,其保留两种复合物的天然寡聚化状态并允许估计其含量。
【背景】类囊体是藻类和植物叶绿体和蓝细菌的特殊膜。类囊体脂质双层与原核细菌膜具有特征性特征,但它富含糖脂,即半乳糖脂,而酸性脂质的水平相对较低,并且不超过总脂质含量的20%。类囊体含有大量蛋白质及其复合物,其中超过40%参与光合作用的光依赖性反应。第二大功能组包括参与蛋白质靶向,加工和折叠,氧化应激反应和翻译的蛋白质。类囊体内最完整的膜蛋白形成大蛋白质复合物,其在光的收获及其光化学转化中起重要作用。执行此转换的关键复合体是PSI和PSII。它们都进行光驱动的初级电荷分离,导致类囊体相关的电子流减少NADP + 并产生用于合成ATP的pH梯度。这两个复合体都经过深入研究,主要集中在阐明它们的功能和潜在的结构特征以及它们的组装机制。对于这项研究,天然膜的分离及其关于两种光系统的含量,组装和低聚状态的详细分析是必不可少的。现有的和经常使用的分离蓝藻类囊体的方案或者不能充分保留脆性膜复合物的天然结构,如光系统II二聚体(例如,Komenda和Barber,1995),或者所获得的制剂含有高浓度的化合物(例如1M甘氨酸甜菜碱)与透明的天然凝胶电泳不相容(例如,Gombos 等。,1994)。从两个观点来看,所描述的类囊体制剂方案都是优化的。使用Wittig 等(2007)修改的方案通过透明原生PAGE(CN PAGE)分析蓝藻膜,提供了关于多亚基色素光系统复合物的数量和寡聚(或组装)状态的直接信息。以前所未有的速度和分辨率膜的分离,溶解和分析不会破坏颜料和蛋白质之间弱的非共价相互作用,因此可以直接扫描具有分离的单个复合物的凝胶。与变性SDS凝胶不同,不需要固定,染色和脱色,或印迹和免疫检测来区分PSII和PSI(参见图2)。绿色PSI复合物在室温下不显示红色荧光,并且在凝胶中作为主要绿色条带存在,而PSII复合物发荧光并且其绿色条带在简单透明度扫描上难以区分。因此,使用绿色非荧光带的简单透明度扫描进行PSI量化,而使用荧光带的红色荧光扫描进行PSII量化。

关键字:蓝藻, 光合作用, 类囊体膜, 光合系统, 非变性聚丙烯酰胺凝胶电泳, 电泳

材料和试剂

  1. 移液器吸头
  2. 2毫升螺旋盖聚丙烯微量瓶(Biospec Products,目录号:3205)
  3. Hamilton ®微量注射器系列700,0.25 ml(Sigma,目录号:24538-U)
  4. Hamilton针,51 mm(Sigma,目录号:21746)
  5. Binder Clip(得力8584,Luma交易)
  6. 磁力搅拌棒,直径5毫米,长20毫米
  7. 透明塑料薄膜
  8. 1.5毫升光谱比色皿(Fisherbrand PS,目录号:7755.0302)
  9. 蓝细菌 Synechocystis sp。的葡萄糖耐受菌株。 PCC6803(GT-P,Tichý等人,2016)
  10. 丙烯酰胺用于分子生物学(AA)(AppliChem Panreac,目录号:A3812,1000)
  11. 用于分子生物学的双丙烯酰胺(BIS)(AppliChem Panreac,目录号:A3636,0250)
  12. 6-氨基己酸(ACA)(AppliChem Panreac,目录号:A2266,500)
  13. Bis-Tris(AppliChem Panreac,产品目录号:A1025.1000) 
  14. MES(AppliChem Panreac,目录号:A0689.0250)
  15. Tricine(AppliChem Panreac,目录号:A1085,1000)
  16. N,N,N',N'-四甲基乙二胺(TEMED)(Sigma,目录号:T928-25ml)
  17. 过硫酸铵(APS)(Sigma,目录号:T3678-25g)
  18. 二硫苏糖醇(DTT)(AppliChem Panreac,目录号:A1101,0025)
  19. SDS(Sigma,目录号:7172-100g)
  20. 直径150-212μm的玻璃珠(Sigma,目录号:G1145)
  21. 正十二烷基-β-D-麦芽糖苷(DM)(AppliChem Panreac,目录号:A0819,0005)
  22. 脱氧胆酸钠(Sigma,目录号:30970-25g)
  23. 用于蛋白质分子量的凝胶过滤标记试剂盒(Sigma,目录号:MWGF1000-1KT)
  24. CaCl 2 (分析级)
  25. MgCl 2 (分析级)
  26. 甲醇(分析级)
  27. 氢氧化钠(分析纯)
  28. 盐酸(分析级)
  29. 甘油(HPLC级)
  30. Milli-Q去离子水(Merck Millipore,Ultrapure [Type 1] water)
  31. 缓冲液B(见食谱)
  32. 凝胶缓冲液6x(pH 7.6)(见食谱)
  33. 丙烯酰胺溶液(AB)(见食谱)
  34. 上部(阴极)缓冲液10x(见食谱)
  35. 上部(阴极)缓冲液1x(见食谱)
  36. 较低(阳极)缓冲液10倍(见食谱)
  37. 下(阳极)缓冲1x(见食谱)
  38. 解析凝胶和堆积凝胶(见食谱)

设备

  1. 自动移液器(P1000,P200和P10)
  2. Mini-beadbeater(西格玛,产品目录号:Z249289)
  3. 磁力搅拌器Color Squid White(Thermo Fisher Scientific,目录号:6110.1005)
  4. GE梯度制造商(Sigma,目录号:GESG50) 
  5. Vortex(Velp Scientifica,型号:RX3)
  6. 离心机(海蒂诗,型号:Micro22 R)
  7. UV-VIS分光光度计(Shimadzu,型号:UV3000)
  8. 冷却微量离心机(Eppendorf,型号:5415R) 
  9. PAGE装置(Bio-Rad,PROTEAN ® II xi Cell)
  10. 电源(Bio-Rad,PowerPac TM HV高压)
  11. 冷却恒温器(Labio,型号:CTB 06C)
  12. LAS 4000相机(富士;叶绿素荧光扫描使用LED蓝光光源460 nm激发,R670滤光片用于发射)
  13. 扫描仪HP ScanJet G4050(HP,型号:G4050)
  14. pH计(赛多利斯,PB-11)
  15. FIM-150片状制冰机(Biobase)

软件

  1. ImageJ与Java 1.8.0_112捆绑在一起( https://imagej.nih.gov/ij/download.html )

程序

  1. 该方案需要在35μmolm -2 s的冷白色荧光灯下,在轨道振荡器(约60rpm)下在30℃下生长的20ml Synechocystis PCC 6803 <液体BG11中的sup> -1 (Rippka et al。,1979),在750nm处光密度为~0.4-0.6(参见注释)。通过在~4℃下在2,300×g离心10分钟收集蓝细菌细胞,将细胞沉淀重悬于1.5ml缓冲液B中,并将重悬浮的细胞转移到2ml螺旋盖微管中。从这一点开始,将管子保持在冰上。
  2. 将离心管在9,300 xg 中于4°C离心2分钟,弃去上清液,将沉淀重悬于0.15 ml缓冲液B中,加入相同体积的玻璃珠(分别测量将0.5ml微管刻度,并加入带有样品的2ml微管中。
  3. 在Mini-Beadbeater中以30秒(最大速度)和1分钟冷却的三个循环打破细胞。
  4. 加入0.2ml缓冲液B,将管涡旋2秒,立即将上清液转移到新的预冷2ml螺旋盖微管中,重复5次。
  5. 将管在400℃下在4℃下离心1分钟以除去未破碎的细胞和玻璃珠。
  6. 将上清液转移到新的预冷却的2ml螺旋盖微量瓶中,并在4℃下以16,000 x g 离心20分钟。
  7. 除去上清液,用0.25 ml微量注射器(长度120 mm,直径2 mm)的不锈钢活塞将沉淀重悬于0.2 ml缓冲液B中:取出上清液后,将颗粒置于颗粒中第二只手,将活塞插入管子直至底部并用插入的活塞涡旋15秒,然后加入0.2毫升缓冲液并再次用活塞内部涡旋10秒。
  8. 测量悬浮液中的叶绿素(Chl)含量。将13.5μl悬浮液与1.5μl10%DM混合,在16,000 xg 离心5分钟,取10μl上清液,加入990μl甲醇螺旋盖小瓶,涡旋,离心机在16,000 xg 处理5分钟,取上清液并测量720和666 nm处的吸光度,C(μgChl/ ml)=(A 666 - A 720 )x 1,269(Wellburn,1994)。
  9. 准备解析凝胶(见视频1)。
    1. 首先根据制造商的说明从玻璃板和1 mm垫片组装凝胶盒。&nbsp;
    2. 然后使用磁力搅拌器和搅拌棒(每种溶液的组分显示在配方8中,一列是一种溶液,第二和第三列),对每种4%和14%的溶解凝胶溶液充分混合所有组分(除了APS!) ;然后在恒定温和搅拌下将APS加入两种溶液中。&nbsp;
    3. 将4%溶液与搅拌棒一起倒入位于磁力搅拌器顶部的梯度制造器的右侧隔室中。梯度制造器具有连接到硅胶管的入口,该硅胶管通向蠕动泵并且具有通向第二隔室的封闭连接通道。蠕动泵的出口使用粘合剂夹连接到与玻璃凝胶盒上部连接的皮下注射针。&nbsp;
    4. 小心地打开连接通道,使其充满4%溶液(如果释放更多溶液,将其移回右侧隔室)并再次关闭,然后将14%溶液添加到第二个(左侧)隔室中渐变制造者。&nbsp;
    5. 用移液管除去8.5毫升14%的溶液,直接加到玻璃盒底部进行凝胶聚合,然后立即开始以每分钟约10毫升的速度将剩余的溶液泵入玻璃盒中并平行打开连接制造商的隔间之间的通道。调节溶液的温和搅拌(每秒2转)。&nbsp;
    6. 倒出梯度后,取下针头,用0.25 ml注射器用0.5 ml蒸馏水将溶液覆盖在凝胶盒中,然后用Milli-Q水清洗梯度制造器和管道。观察水和4%溶液之间的界面,直到形成清晰的边界,然后用注射器除去水。&nbsp;

    视频1。浇注解析凝胶

  10. 准备堆积凝胶。将APS加入10 ml 4%溶液(配方8中的右栏)中,倒入聚合的溶解凝胶顶部,浸入10孔梳子,避免产生气泡,等待聚合反应;然后取出梳子并用Milli-Q水清洗孔。
  11. 将准备好的凝胶连接到冷却芯中,检查连接是否紧密并且没有用蒸馏水泄漏,倒出水并将带有凝胶的核心插入带有2升1x低缓冲液的电泳室中,连接冷却的硅管恒温器的核心,设定为4°C,并将200毫升的1x上部缓冲液倒入冷却核心的上部储液器中。
  12. 对于凝胶分析,取含有5μgChl的样品,加入相应体积的缓冲液B缓冲液以使所有样品达到相同的体积(通常为30μl),加入1/10体积的10%DM并立即以16,000 <离心em> xg 在4°C下保持20分钟。
  13. 取25μl溶解的膜并加载到凝胶孔中。
  14. 为了估计复合物的分子大小,可以使用蛋白质分子量的凝胶过滤标记试剂盒,加载30μl推荐的混合物。
  15. 关闭盖子,将其连接到电源并以15 mA的恒定电流模式运行1.5-2小时,电压上限为1,000 V(参见图1;运行电压逐渐增加至1,000 V,之后达到这个极限,电流下降了。


    图1.带冷却恒温器(左)和电源(右上角)的电泳装置(右下角)的设置

  16. 在着色的前部距离井底部约8cm的距离后,关闭电源,取下盖子,拆下硅树脂冷却管并用电泳室中的凝胶取出冷却芯。
  17. 从玻璃盒中取出凝胶并将其包裹在透明塑料薄膜中。
  18. 将其传输到扫描仪中以获取彩色图像(300 dpi分辨率,48位颜色深度)。
  19. 将凝胶转移到LAS4000相机中,以透明模式拍摄tiff格式的16位灰度图像,以量化PSI的绿色条带(数字化DIA方法,最低托盘位置,3秒曝光,高分辨率模式)或使用落射荧光模式蓝色LED激发光(波长460 nm)引发叶绿素(但不是藻胆蛋白)荧光,同时发出的光通过R670滤光片(边长为670 nm的长通滤光片)定量PSII(荧光法,最低的托盘)位置,3秒曝光,高分辨率模式)(见图2)。


    图2.凝胶的代表性彩色扫描(左对)和叶绿素荧光图像(右对),分离的光系统I和来自蓝细菌的光系统II复合物 Synechocystis PCC 6803 GT-P (Tichý et al。,2016)和光系统I-less突变体(Shen 左边的两个泳道显示彩色扫描,右边的荧光扫描用光系统II二聚体和单体的显性条带进行扫描,右边的一个荧光扫描。,1993)。蓝色条带(PBS frag。)代表藻胆体的片段,蓝藻天线,其荧光不会被蓝光引发。

  20. 根据用户指南使用ImageJ量化波段( https://imagej.nih .GOV / IJ /文档/导向/用户指南.pdf )。首先使用工具栏工具中的区域选择工具选择波段作为感兴趣区域(ROI),使用处理工具减去背景,并使用分析工具中的强度统计分析波段的强度。

笔记

除了蓝藻 Synechocystis PCC 6803之外,所述方案已经成功地测试了其他蓝藻菌株,例如 Synechococcus elongatus PCC 7942和 Synechococcus sp。 PCC 7002.对于绿藻 Chlamllaomonas reinhardtii , Chlorella sorokiniana 和 Scenedesmus quadricauda 方案相同,只有较大的玻璃珠直径为0.5 mm(Sigma)应该用于破坏细胞。 CN PAGE的程序也可用于来自植物叶绿体的分离的类囊体膜。

食谱

  1. 缓冲液B(50毫升库存)
    25mM MES / NaOH缓冲液(pH6.5)
    10mM CaCl 2
    10mM MgCl 2
    25%甘油
    分成10毫升等分试样并在-25°C冷冻保存(稳定至少6个月)
  2. 凝胶缓冲液6x(pH7.0,100ml)
    300 mM Bis-Tris / HCl缓冲液
    用4 M HCl调节至pH 7.0 储存在4°C(4°C下至少稳定6个月)
    注意:通常准备100毫升。
  3. 丙烯酰胺溶液(AB)
    50%AA
    1.33%BIS
    Milli-Q水
    储存在4°C(稳定至少3个月)
    注意:通常准备500毫升。
  4. 上部(阴极)缓冲器10x
    0.5 M tricine
    150 mM Bis-Tris / HCl
    使用4 M HCl将pH调节至7.0
    储存在4°C(4°C下至少稳定6个月)
    注意:通常准备500毫升。
  5. 上部(阴极)缓冲液1x(一页200毫升)
    1. 将20毫升上层缓冲液10倍加入180毫升Milli-Q水和1毫升10%脱氧胆酸钠中,使其达到0.05%终浓度
    2. 加入0.4ml 10%DM至终浓度0.02%。
    注意:在跑步前准备新鲜的。
  6. 下(阳极)缓冲10x
    0.5 M Bis-Tris / HCl
    用4 M HCl将pH调节至7.0
    储存在4°C(4°C下至少稳定6个月)
    注意:通常准备500毫升。
  7. 下(阳极)缓冲器1x(2 L)
    用Milli-Q水将下部(阳极)缓冲液稀释10倍至较低(阳极)缓冲液1x
    注意:此溶液在4°C下稳定至少3个月,可以重复使用三次。
  8. 解析凝胶和堆积凝胶
    混合各个组分(左起第一列)得到4%(第二列)和14%(第三列)溶液用于制备分离梯度凝胶和4%溶液用于制备堆积凝胶(第四列)到下表:

致谢

这项工作得到了捷克共和国捷克教育,青年和体育部(LO1416项目)以及捷克共和国和匈牙利科学院之间的双边项目(MTA-16-11)的支持。

利益争夺

我们声明没有利益冲突。

参考

  1. Gombos,Z。,Wada,H。和Murata,N。(1994)。 膜脂质的不饱和度加速了低温光抑制对光合作用的恢复:一种机制令人不寒而栗。 Proc Natl Acad Sci USA 91(19):8787-8791。
  2. Komenda,J。和Barber,J。(1995)。 Synechocystis PCC 6803的psbO和psbH缺失突变体的比较表明降解D1蛋白受QB位点调节并依赖于蛋白质合成。 Biochemistry 34(29):9625-9631。
  3. Rippka,R.,Deruelles,J.,Waterbury,J.B.,Herdman,M。和Stanier,R。Y.(1979)。 蓝细菌纯培养物的一般分配,菌株历史和特性。 微生物学 111:1-61。
  4. Shen,G.,Boussiba,S。和Vermaas,W。F.(1993)。 Synechocystis sp PCC 6803菌株缺乏光系统I和藻胆体功能。 植物细胞 5(12):1853-1863。
  5. Tichý,M.,Beckova,M.,Kopecna,J.,Noda,J.,Sobotka,R。和Komenda,J。(2016)。 Synechocystis 的菌株 PCC 6803与光系统II的异常组装包含串联重复大型染色体区域。 Front Plant Sci 7:648。
  6. Wellburn,A.R。(1994)。 叶绿素a和b以及总类胡萝卜素的光谱测定,使用各种溶剂和分光光度计 不同的分辨率。 J Plant Physiol 144(3):307-313。
  7. Wittig,I.,Karas,M。和Schagger,H。(2007)。 高分辨率透明天然电泳,用于凝胶功能测定和膜蛋白复合物的荧光研究。 Mol Cell Proteomics 6(7):1215-1225。
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Copyright: © 2019 The Authors; exclusive licensee Bio-protocol LLC.
引用:Komenda, J., Krynická, V. and Zakar, T. (2019). Isolation of Thylakoid Membranes from the Cyanobacterium Synechocystis sp. PCC 6803 and Analysis of Their Photosynthetic Pigment-protein Complexes by Clear Native-PAGE. Bio-protocol 9(1): e3126. DOI: 10.21769/BioProtoc.3126.
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