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Analysis of Starch Synthase Activities in Wheat Grains using Native-PAGE
采用非变性聚丙烯酰胺凝胶电泳分析麦粒中淀粉合成酶的活性   

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Abstract

Starch synthases are one class of key enzymes involving in the synthesis of cereal starch, which transfer glucose from ADP-glucose to the non-reducing end of pre-existing α-(1-4)-liked glucosyl chains of amylopectin. This protocol is highly reproducible for assaying activities for starch synthase I and IIIa in wheat and barley endosperm at qualitative level and quantitative level. The protocol includes separating proteins isolated from developing endosperm with native-PAGE containing glycogen from oyster, incubating protein gels with ADP-glucose solution, and staining gels with iodine solution. The method allows researchers to compare the levels or changes of starch synthase activities.

Keywords: Starch synthase(淀粉合成酶), Enzymatic activity assay(酶活性测定), Wheat(小麦), Grain(粮食), Native-PAGE(本地页面)

Materials and Reagents

  1. Glad-wrap (Microwave safe, Capri)
  2. Kimwipe (Kimtech Science* Kimwipes delicate task wipers) (Kimberly-Clark, catalog number: 34133 )
  3. Gel-cassette (size of 100 mm x 100 mm with 1 mm gap) (Invitrogen, catalog number: NC2010 )
    Note: Currently, it is “Thermo Fisher Scientific, Novex™, catalog number: NC2010”.
  4. 15 ml blue cap Falcon tube (sterile) (Thermo Fisher Scientific)
  5. Epipestle (bioWORLD, catalog number: 42741000-1 )
  6. 96 well UV microplate (Flat bottom) (Thermo Fisher Scientific, catalog number: 8404 )
  7. Cuvette (plastic) (SARSTEDT AG & Co, catalog number: 67.746 )
  8. MilliQ water
  9. 70% ethanol (Chem Supply, catalog number: 64-17-5 )
  10. 2% agarose (Progen, catalog number: 200-0011 )
  11. Tris (2-Amino-2-hydroxymethyl-propane-1, 3-diol) (VWR International, catalog number: 103157P )
  12. Glycogen from oyster (Sigma-Aldrich, catalog number: G8751 )
  13. Acrylamide (40% Acrylamide/Bis Solution, 37.5:1) (Bio-Rad Laboratories, catalog number: 161-0148 )
  14. TEMED (Tetramethylethylenediamine) (AMRESCO, catalog number: 0761-25 ML )
  15. APS (Ammonia persulfate) (Sigma-Aldrich, catalog number: A-7460 )
  16. Coomassie Plus Protein Assay Reagent (Thermo Fisher Scientific, catalog number: 1856210)
  17. Bovine serum albumin (BSA) (Freeze dried, Reagent Grad, pH 7) (Moregate Biotech)
  18. HCl (Ajax Finechem Pty, catalog number: 1367-2.5 L )
  19. Ammonium Sulfate [(NH4)2SO4] (AR) (Chem Supply, catalog number: AA014-500 G )
  20. Magnesium Chloride Hexahydrate (MgCl2) (AR) (Chem Supply, catalog number: MA029-500 G )
  21. β-mercaptoethanol (Sigma-Aldrich, catalog number: M-7154 )
  22. Adenosine-5’-diphosphoglucose disodium salt (ADPG) (Sigma-Aldrich, catalog number: A0627-250 mg )
  23. Protease inhibitor cocktail for plant cell and tissue extracts (Sigma-Aldrich, catalog number: P9599 )
  24. DL-Dithiothreitol (DTT) (Sigma-Aldrich, catalog number: D-9779 )
  25. Glycine (Chem Supply, catalog number: GA007-500 G )
  26. Bromophenol blue (Sigma-Aldrich, catalog number: B0126-25 G )
  27. Glycerol (AR) (Chem Supply, catalog number: GA010-2.5 L )
  28. Extraction buffer (see Recipes)
  29. 1x electrophoresis buffer (see Recipes)

Equipment

  1. Gel tank (XCell SureLock) (Invitrogen, catalog number: EI0001 )
    Note: Currently, it is “Thermo Fisher Scientific, NovexTM, catalog number: EI0001”.
  2. Pipette (Thermo Fisher Scientific, model: Finnpipette F1 )
  3. Spectrophotometer (Agilent Technologies, model: Cary 300 Bio UV-Visible Spectrophotometer )
  4. Microplate reader (BMG LABTECH GmbH, model: FLUOstar Omega Microplate Reader )

Procedure

  1. Prepare 8% Native-PAGE gels
    1. Preparing gel cassettes
      Both commercial cassettes and self assembled cassettes with glass plates can be used. If not using commercial cassettes, glass plates, 1 mm spacers and combs need to be thoroughly cleaned with MilliQ water and then with 70% ethanol (use of 1 mm gels gives better resolution than 1.5 mm gels). Glass plates need to be secured with clamps - don’t take clamps past inside of spacers. The bottom of plates is sealed by standing plates in molten 1 ml of 2% agarose in 0.3 M Tris/HCl, pH 8.8 poured onto Glad-wrap.
    2. Preparing separating gel layer (8% Native-PAGE) as Table 1
      1. First dissolve 34.5 mg glycogen from oyster in 3.473 ml water (for 1 gel) or 69 mg glycogen in 6.945 ml water (for 2 gels) in a 15 ml blue cap Falcon tube.
      2. Add 1 M Tris/HCl, pH 8.8 and 40% acrylamide (37.5:1).
      3. Degas the gel solution for 5 min to remove air using water tap vacuum.
      4. Add TEMED and 10% APS.

        Table 1. Chemicals and solutions for separating gels

        1 gel (7.5 ml)
        2 gels (15 ml)
        Glycogen from oyster
        34.5 mg
        69 mg
        H2O
        3.473 ml
        6.945 ml
        1 M Tris/HCl, pH 8.8
        2.475 ml
        4.95 ml
        40% acrylamide (37.5:1)
        1.5 ml
        3 ml
        Add items below last for polymerizing gel




        TEMED
        7.5 μl
        15 μl
        10% APS
        45 μl
        90 μl

        Notes:
        1. *Tips and tubes used for acrylamide solution need to be discarded in the acrylamide waste bin for toxic waste management.
        2. 10% APS: Make 500 μl solution and aliquot to 65 μl per tube and store at -20 °C. Take one tube out of -20 °C freezer each time to use. The same tube should only be used for 1 week after thawing and storing at 4 °C. Discard the tube more than 1 week after thawing.

      5. Pour the gel mix into the cassette to 1-1.5 cm below the bottom of the comb using 1 ml pipette. Add 0.5 ml of isopropanol on the top of the gel mix inside of the cassette to level the top of the gel and to aid in the gel polymerisation. Leave the gel mix for polymerisation for ~30 min at room temperature.
      6. Before adding stacking gel layer, pour off isopropanol and wash at least twice with MilliQ H2O. Carefully wipe MilliQ H2O between the two plates with a Kimwipe to remove as much excess MilliQ H2O as possible without touching the gel.

    3. Preparing stacking gel layer
      1. Make stacking gel mix in a 15 ml blue cap Falcon tube as Table 2.

        Table 2. Solutions for stacking gels

        1 gel (2.5 ml)
        2 gels (5 ml)
        0.5 M Tris/HCl (pH 6.8)
        650 μl
        1.3 ml
        40% acrylamide (37.5:1)
        285 μl
        570 μl
        H2O
        1.5475 ml
        3.095 ml
        Add items below last for polymerizing gel




        TEMED
        2.5 μl
        5 μl
        10% APS
        15 μl
        30 μl

      2. Pour the stacking gel into the cassette and add a comb. If pouring more than one gel, add the comb to the stacking gel of the first gel poured before pouring the second gel. Leave the stacking gel mix for another ~30 min for polymerization.
      3. If it has been planned to run the gel the next day, the gel needs to be covered with Glad-wrap and stored at 4 °C.

  2. Prepare protein samples
    1. Protein extraction
      The following steps for protein extraction need to be processed on ice or 4 °C to avoid protein degradation.
      1. Take 1-2 developing endosperms (12 to 15 days after anthesis) and place in an Eppendorf tube, and then weigh.
      2. Add 100 μl of extraction buffer (see Recipes) per 100 mg tissue.
      3. Grind seeds to a pulp using an epipestle, spin at 14,000 x g for 15 min at 4 °C.
      4. Transfer supernatant into a new Eppendorf tube.
      5. If sediment materials are mixed with supernatant, spin for 5-10 min at 4 °C again and transfer supernatant into another new Eppendorf tube.
    2. Protein quantification
      1. Prepare a protein concentration standard curve
        1. Prepare BSA solution (0.25 mg/ml) by diluting BSA solution (1 mg/ml) in a ratio of 1:4 with MilliQ H2O.
        2. Construct a standard curve for protein concentration using BSA (0.25 mg/ml) as in Table 3 for 1 ml cuvette or Table 4 for 96 well UV microplate.

          Table 3. Preparation of protein standards for 1 ml cuvette
          Protein concentration (μg/1,100 μl)
          BSA volume (μl)
          H2O (μl)
          Coommassie plus protein assay reagent (μl)
          0
          0
          100
          900
          5
          20
          80
          900
          10
          40
          60
          900
          15
          60
          40
          900
          20
          80
          20
          900
          25
          100
          0
          900

          Table 4. Preparation of protein standards for 96 well UV microplate
          Protein concentration (μg/220 μl)
          BSA volume (μl)
          H2O (μl)
          Coommassie plus protein assay reagent (μl)
          0
          0
          20
          180
          5
          4
          16
          180
          10
          8
          12
          180
          15
          12
          8
          180
          20
          16
          4
          180
          25
          20
          0
          180

      2. Preparation of samples
        For 1 ml cuvette, add 3 μl of sample to 97 μl H2O and 900 μl of Coomassie Plus Protein Assay Reagent.
        For 96 well plate, add 1 μl (or less) of sample to 19 μl H2O (or more) and 180 μl of Coomassie Plus Protein Assay Reagent.
      3. Measure the protein absorbance with a spectrophotometer for cuvettes or a microplate reader for 96 well UV microplate
        1. Assay protein standards and samples on a spectrophotometer or a microplate reader.
        2. Read absorbance at 595 nm.
        3. Match absorbance against a standard curve. Divide μg in sample by number of μl added, in this case 3, to give final concentration in μg/μl.
        4. Check that color falls within color range of standards. If not, reduce or increase amount of sample accordingly.
        5. Store protein samples by freezing samples in liquid nitrogen and storing at -80 °C.

    Notes:
    1. Read absorbance for protein standards and samples at the same time.
    2. Construct protein standard curve in Excel as Figure 1:
      1. Construct scatter chart.
      2. Select data in the Excel sheet, click “Insert” tab, then “Charts” tab, click “Scatter” icon, and add “Trendline”.
      3. Under the Type tab, select “Linear”.
      4. Check “Set Intercept = 0” and check “Display equation on chart” and “Display R-squared value on chart”.
      5. Use the equation to determine protein content of samples (Don’t forget to divide by the number of μl of sample added to give concentration in μg/μl).


        Figure 1. A standard curve for measuring protein concentration using BSA as protein standard. The X-axis is the protein concentration and the Y-axis is the absorbance measured at 595 nm.

  3. Gel electrophoresis
    1. Assemble gels.
    2. Pour 800 ml 1x electrophoresis buffer into gel tank.
    3. Add the loading buffer to protein sample solution on ice.
      Perform following steps in fume hood:
      To each 100 μl of protein sample solution, add:
      10 μl 0.5 M Tris-HCl (pH 6.8)
      10 μl β-mercaptoethanol
      5 μl 0.6% bromophenol blue in 80% glycerol
    4. Re-calculate concentration to allow for addition of above denaturing buffer to proteins. New final concentration= (initial volume/final volume) x initial concentration.
    5. Load 100 μg total protein for each sample in each well.
    6. If space permits, leave a lane between ladder and samples.
    7. Run gels at a maximum voltage at 100 V (or a maximum ampere at 15 mA per gel).
    8. Running gels until the bromophenol blue dye reaches the bottom of the gel.

  4. Assay starch synthase activity
    1. Prepare starch synthase assay buffer as Table 5.

      Table 5. Starch synthase assay buffer

      10 ml
      15 ml
      Note
      10x Tris-Glycine
      (10x running buffer)
      1 ml
      1.5 ml

      2 M (NH4)2SO4
      667 μl
      1 ml

      2 M MgCl2
      37μl
      55 μl

      BSA (dried)
      6.7 mg (0.0067 g)
      10 mg (0.010 g)

      β-mercaptoethanol
      47 μl
      70 μl

      360 mM ADPG (-20 °C)
      34 μl
      51 μl
      Add last as it goes off quickly
      H2O
      8.22 ml
      12.32 ml


      2 M MgCl2
      2 M (NH4)2SO4
      MW=203.3 g
      2 M= 4.066 g/10 ml
      MW= 132.13 g
      2 M= 2.643 g/10 ml

      Note: 360 mM ADPG needs to be kept as 34 μl aliquot and kept at -20 °C. Take out one tube for each gel from freezer before use.

    2. Incubate the gel(s) with starch synthase assay buffer overnight at room temperature (~22 °C).
    3. Wash gel(s) two times with MilliQ H2O.
    4. Stain gel(s) with iodine solution (containing 2% KI, 0.2% I2) until brown bands appear as Figure 2, usually about 10 min. Change iodine solution after 10 min if necessary.
    5. The gels can be kept in iodine solution if needed.

Representative data

  1. Representative examples of starch synthase activity results can be expected in barley grain as Figure 2(b) (from Figure 8A, Li et al., 2011) and in wheat grain as Figure 2(c) (From Figure 2, McMaugh et al., 2014).


    Figure 2. Starch synthase activity of developing endosperm of barley and wheat. (a). A native-PAGE gel before staining with iodine solution. (b). A native-PAGE gel showing starch synthase I and IIIa activities in barley developing endosperm after staining with iodine solution. (c). A native-PAGE gel showing starch synthase I and IIIa activities in wheat developing endosperm after staining with iodine solution.

  2. This protocol is highly reproducible in our hand. The quality of native polyacrylamide gels determines the quality of protein bands. The quality of both TEMED and APS may affect the gel quality and then protein band quality.

Notes

  1. Glycogen from oyster is the best substrate for native polyacrylamide gel (native-PAGE).

Recipes

  1. Extraction buffer
    Add 20 μl of 1 M potassium phosphate buffer, pH 7.5 (final concentration: 50 mM KPi)
    Add 1 μl of 500 mM EDTA, pH 7.5 (final concentration: 5 mM EDTA)
    Add 400 μl of 50% glycerol (final concentration: 20% glycerol)
    Add 567 μl MilliQ H2O
    Add Protease inhibitor cocktail for plant cell and tissue extracts and DTT to buffer as below when ready to extract
    Add 2 μl Protease inhibitor cocktail for plant cell and tissue extracts
    Add 10 μl of 500 mM DTT (final concentration: 5 mM DTT)
  2. 1x electrophoresis buffer
    Add 80 ml 10x running buffer
    Add DTT at 0.15 g /L
    Make up to 800 ml in distilled water
    10x running buffer (10x Tris-glycine Stock Solution)
    1.9 M glycine (144 g/L)
    250 mM Tris (30.3 g/L)

Acknowledgments

This protocol was modified from previous publication (Abel et al., 1996). The author thanks Behjat Kosar-Hashemi, Emma Anschaw, Steve McMaugh, Sapna Vibhakaran Pillai and Hong Wang for their contributions in optimizing and testing this protocol. The author also thanks CSIRO Plant Industry, CSIRO Food Future Flagship and ACVL Ltd for providing funding resource for testing this protocol during the course of research.

References

  1. Abel, G. J., Springer, F., Willmitzer, L. and Kossmann, J. (1996). Cloning and functional analysis of a cDNA encoding a novel 139 kDa starch synthase from potato (Solanum tuberosum L.). Plant J 10(6): 981-991.
  2. Li, Z., Mouille, G., Kosar-Hashemi, B., Rahman, S., Clarke, B., Gale, K. R., Appels, R. and Morell, M. K. (2000). The structure and expression of the wheat starch synthase III gene. Motifs in the expressed gene define the lineage of the starch synthase III gene family. Plant Physiol 123(2): 613-624.
  3. Li, Z., Li, D., Du, X., Wang, H., Larroque, O., Jenkins, C. L., Jobling, S. A. and Morell, M. K. (2011). The barley amo1 locus is tightly linked to the starch synthase IIIa gene and negatively regulates expression of granule-bound starch synthetic genes. J Exp Bot 62(14): 5217-5231.
  4. McMaugh, S. J., Thistleton, J. L., Anschaw, E., Luo, J., Konik-Rose, C., Wang, H., Huang, M., Larroque, O., Regina, A., Jobling, S. A., Morell, M. K. and Li, Z. (2014). Suppression of starch synthase I expression affects the granule morphology and granule size and fine structure of starch in wheat endosperm. J Exp Bot 65(8): 2189-2201.

简介

淀粉合酶是涉及谷物淀粉合成的一类关键酶,其将葡萄糖从ADP-葡萄糖转移到预先存在的支链淀粉的α-(1-4) - 葡萄糖基链的非还原端。 这个协议是高度可重复的测定淀粉合成酶I和IIIa在小麦和大麦胚乳的定性水平和定量水平的活动。 该方案包括从发育的胚乳分离的蛋白质与来自牡蛎的含有糖原的天然PAGE,用ADP-葡萄糖溶液孵育蛋白质凝胶,并用碘溶液染色凝胶。 该方法允许研究人员比较淀粉合酶活性的水平或变化。

关键字:淀粉合成酶, 酶活性测定, 小麦, 粮食, 本地页面

材料和试剂

  1. 高兴包装(微波保险箱,Capri)
  2. Kimwipe(Kimtech Science * Kimwipes精致任务擦拭器)(Kimberly-Clark,目录号:34133)
  3. 凝胶盒(尺寸为100mm×100mm,1mm间隙)(Invitrogen,目录号:NC2010) 注意:目前,它是"Thermo Fisher Scientific,Novex?,目录号:NC2010"。
  4. 15ml蓝帽Falcon管(无菌)(Thermo Fisher Scientific)
  5. Epipestle(bioWORLD,目录号:42741000-1)
  6. 96孔UV微孔板(平底)(Thermo Fisher Scientific,目录号:8404)
  7. 比色杯(塑料)(SARSTEDT AG& Co,目录号:67.746)
  8. MilliQ水
  9. 70%乙醇(Chem Supply,目录号:64-17-5)
  10. 2%琼脂糖(Progen,目录号:200-0011)
  11. 三(2-氨基-2-羟甲基 - 丙烷-1,3-二醇)(VWR International,目录号:103157P)
  12. 来自牡蛎的糖原(Sigma-Aldrich,目录号:G8751)
  13. 丙烯酰胺(40%丙烯酰胺/Bis溶液,37.5:1)(Bio-Rad Laboratories,目录号:161-0148)
  14. TEMED(四甲基乙二胺)(AMRESCO,目录号:0761-25ML)
  15. APS(过硫酸铵)(Sigma-Aldrich,目录号:A-7460)
  16. Coomassie Plus Protein Assay Reagent(Thermo Fisher Scientific,目录号:1856210)
  17. 牛血清白蛋白(BSA)(冻干,Reagent Grad,pH 7)(Moregate Biotech)
  18. HCl(Ajax Finechem Pty,目录号:1367-2.5L)
  19. 硫酸铵[(NH 4)2 SO 4](AR)(Chem Supply,目录号:AA014-500G)
  20. 六水合氯化镁(MgCl 2)(AR)(Chem Supply,目录号:MA029-500G)
  21. β-巯基乙醇(Sigma-Aldrich,目录号:M-7154)
  22. 腺苷-5'-二磷酸葡萄糖二钠盐(ADPG)(Sigma-Aldrich,目录号:A0627-250mg)
  23. 用于植物细胞和组织提取物的蛋白酶抑制剂混合物(Sigma-Aldrich,目录号:P9599)
  24. DL-二硫苏糖醇(DTT)(Sigma-Aldrich,目录号:D-9779)
  25. 甘氨酸(Chem Supply,目录号:GA007-500G)
  26. 溴酚蓝(Sigma-Aldrich,目录号:B0126-25G)
  27. 甘油(AR)(Chem Supply,目录号:GA010-2.5L)
  28. 提取缓冲液(参见配方)
  29. 1x电泳缓冲液(参见配方)

设备

  1. 凝胶罐(XCell SureLock)(Invitrogen,目录号:EI0001) 注意:目前,"Thermo Fisher Scientific,Novex TM ,目录号:EI0001"。
  2. 移液管(Thermo Fisher Scientific,型号:Finnpipette F1)
  3. 分光光度计(Agilent Technologies,型号:Cary 300 Bio UV-Visible分光光度计)
  4. 酶标仪(BMG LABTECH GmbH,型号:FLUOstar Omega Microplate Reader)

程序

  1. 准备8%Native-PAGE凝胶
    1. 准备胶盒
      两个商业盒和自组装 ?可以使用具有玻璃板的盒。如果不使用商业 盒,玻璃板,1毫米间隔和梳子需要彻底 用MilliQ水然后用70%乙醇洗涤(使用1mm凝胶 得到比1.5mm凝胶更好的分辨率)。玻璃板需要 用夹具固定 - 不要夹紧夹具经过间隔垫片内部。的 通过静置板将板的底部密封在熔融的1ml 2% 琼脂糖在0.3M Tris/HCl,pH8.8中的溶液倒在Glad包装上
    2. 准备如表1所示的分离凝胶层(8%Native-PAGE)
      1. 首先从3.763毫升水中的牡蛎中溶解34.5毫克糖原(对于1 ?凝胶)或69mg糖原在6.945ml水中(对于2个凝胶)在15ml蓝色中 盖猎鹰管。
      2. 加入1M Tris/HCl,pH8.8和40%丙烯酰胺(37.5:1)
      3. 使凝胶溶液脱气5分钟,使用水龙头真空除去空气
      4. 添加TEMED和10%APS
        表1.用于分离凝胶的化学品和解决方案

        1凝胶(7.5ml) 2凝胶(15ml)
        牡蛎糖原
        34.5 mg
        69毫克
        H sub 2 O
        3.473 ml
        6.945 ml
        1M Tris/HCl,pH 8.8
        2.475 ml
        4.95 ml
        40%丙烯酰胺(37.5:1) 1.5 ml
        3 ml
        添加下面的项目聚合凝胶




        TEMED
        7.5μl
        15微升
        10%APS
        45微升
        90微升

        注意:
        1. 用于丙烯酰胺溶液的提示和管子需要在丙烯酰胺废物箱中丢弃,以进行有毒废物管理。
        2. <10%APS:制备500μl溶液,并等分至65μl每管和 储存于-20°C。从一个管子中取出-20°C的冷冻机,每次使用。 同一管只应在解冻和储存后使用1周 在4℃。解冻后超过1周后丢弃管。

      5. 将凝胶混合物倒入盒中,低于底部1-1.5厘米 梳使用1ml移液管。在凝胶顶部加入0.5ml异丙醇 ?混合盒内部以平整凝胶的顶部并帮助 凝胶聚合。离开凝胶混合物聚合约30分钟 ?在室温下
      6. 在添加堆叠凝胶层之前,倒入 并用MilliQ H 2 O洗涤至少两次。小心擦拭 MilliQ H sub 2 O在两个板之间用Kimwipe去除尽可能多的 过量的MilliQ H 2 O尽可能不接触凝胶。

    3. 准备堆叠凝胶层
      1. 使堆叠的凝胶混合在15ml蓝帽Falcon管中,如表2所示
        表2.堆叠凝胶的解决方案

        1凝胶(2.5ml) 2凝胶(5ml)
        0.5 M Tris/HCl(pH 6.8)
        650微升
        1.3 ml
        40%丙烯酰胺(37.5:1) 285μl
        570微升
        H sub 2 O
        1.5475 ml
        3.095 ml
        添加下面的项目聚合凝胶




        TEMED
        2.5μl
        5微升
        10%APS
        15微升
        30微升

      2. 将堆叠的凝胶倒入盒中并加入梳子。如果浇注 多于一个凝胶,将梳子添加到第一凝胶的堆叠凝胶 倾倒前倒第二凝胶。留下堆叠胶混合物 另一个?30分钟聚合
      3. 如果计划在第二天运行凝胶,凝胶需要用Glad-wrap覆盖并在4℃下储存。

  2. 准备蛋白质样品
    1. 蛋白质提取
      以下蛋白质提取步骤需要在冰上或4℃下处理以避免蛋白质降解。
      1. 取1-2发育内胚乳(开花后12至15天),并置于Eppendorf管中,然后称重。
      2. 每100 mg组织加入100μl提取缓冲液(见配方)。
      3. 使用epipestle将种子研磨成浆,在4℃下以14,000×g旋转15分钟。
      4. 将上清液转移到新的Eppendorf管中
      5. 如果沉淀物与上清液混合,旋转5-10分钟 再次在4℃并将上清液转移到另一个新的Eppendorf管中。
    2. 蛋白质定量
      1. 准备蛋白质浓度标准曲线
        1. 通过用MilliQ H 2 O以1:4的比例稀释BSA溶液(1mg/ml)制备BSA溶液(0.25mg/ml)。
        2. 使用BSA构建蛋白质浓度的标准曲线 (0.25mg/ml),如表3中对于1ml比色皿或表4对于96孔UV 微孔板
          表3.制备1 ml比色皿的蛋白质标准品
          蛋白质浓度(μg/1,100μl)
          BSA体积(μl)
          H <2 O(μl)
          Coommassie加蛋白测定试剂(μl)
          0
          0
          100
          900
          5
          20
          80
          900
          10
          40
          60
          900
          15
          60
          40
          900
          20
          80
          20
          900
          25
          100
          0
          900

          表4. 96孔紫外微孔板的蛋白标准品的制备
          蛋白浓度(μg/220μl)
          BSA体积(μl)
          H <2 O(μl)
          Coommassie加蛋白测定试剂(μl)
          0
          0
          20
          180
          5
          4
          16
          180
          10
          8
          12
          180
          15
          12
          8
          180
          20
          16
          4
          180
          25
          20
          0
          180

      2. 样品的制备
        对于1ml比色皿,将3μl样品加入到97μlH 2 O和900μlCoomassie Plus蛋白测定试剂中。
        对于96孔板,向19μlH 2 O(或更多)和180μlCoomassie Plus蛋白测定试剂中加入1μl(或更少)的样品。
      3. 用比色杯分光光度计或96孔UV微孔板微孔板读数器测量蛋白质吸光度
        1. 在分光光度计或酶标仪上测定蛋白质标准品和样品
        2. 读取595 nm处的吸光度。
        3. 使吸光度与标准曲线匹配。将样品中的μg除以 ?加入的μl数,在这种情况下为3,得到最终浓度 μg/μl
        4. 检查颜色是否在标准的颜色范围内。如果没有,相应减少或增加样品量
        5. 通过将样品在液氮中冷冻并在-80℃下储存来储存蛋白质样品
    注意:
    1. 同时读取蛋白质标准品和样品的吸光度。
    2. 在Excel中构建蛋白质标准曲线,如图1所示:
      1. 构造散点图。
      2. 在Excel工作表中选择资料,按一下[插入]标签,然后按一下[图表]标签,再按一下[散布]图示,然后新增「趋势线」。
      3. 在类型选项卡下,选择"线性"。
      4. 选中"设置截距= 0"并选中"在图表上显示方程"和"在图表上显示R平方值"。
      5. 使用方程式确定样品的蛋白质含量(不要 忘记除以加入的样品的μl数 浓度(μg/μl)。


        图1.测量的标准曲线 蛋白质浓度,使用BSA作为蛋白质标准品。 X轴是 蛋白质浓度,Y轴是在595测量的吸光度 nm。

  3. 凝胶电泳
    1. 装配凝胶。
    2. 将800 ml 1x电泳缓冲液倒入凝胶罐中。
    3. 在冰上向蛋白质样品溶液中加入上样缓冲液 在通风橱中执行以下步骤:
      向每100μl蛋白质样品溶液中加入:
      10μl0.5M Tris-HCl(pH 6.8)
      10μlβ-巯基乙醇 5μl0.6%溴酚蓝在80%甘油中
    4. 重新计算浓度以允许添加上述变性 ?缓冲液到蛋白质。新的最终浓度=(初始体积/最终 体积)×初始浓度
    5. 每个样品加入100μg总蛋白。
    6. 如果空间允许,在梯子和样品之间留出一条通道
    7. 在100 V(或最大安培,每个凝胶15 mA)的最大电压下运行凝胶。
    8. 运行凝胶,直到溴酚蓝染料到达凝胶底部

  4. 测定淀粉合酶活性
    1. 制备淀粉合酶测定缓冲液,如表5所示
      表5.淀粉合酶测定缓冲液

      10 ml
      15 ml
      注意
      10x Tris-甘氨酸
      (10x运行缓冲区)
      1 ml
      1.5 ml

      2 M(NH 4)2 SO 4 4
      667微升
      1 ml

      2 M MgCl 2
      37微升
      55微升

      BSA(干燥)
      6.7mg(0.0067g) 10mg(0.010g)
      β-巯基乙醇 47μl
      70微升

      360 mM ADPG(-20°C)
      34微升
      51微升
      添加最后一个,因为它很快熄灭
      H sub 2 O
      8.22 ml
      12.32毫升


      2 M MgCl 2
      2 M(NH 4)2 SO 4 4
      MW = 203.3g
      2 M = 4.066g/10ml
      MW = 132.13g
      2 M = 2.643g/10ml

      注意:360 mM ADPG需要保持为34μl等分试样,并保存在-20°C。在使用之前,从冷冻箱中取出一根凝胶。

    2. 在室温(?22℃)下将凝胶与淀粉合酶测定缓冲液孵育过夜
    3. 用MilliQ H 2 O洗涤凝胶两次。
    4. 用碘溶液(含2%KI,0.2%I 2)染色凝胶, 棕色带出现如图2,通常约10分钟。更换碘 溶液,如有必要,10分钟后
    5. 如果需要,凝胶可以保持在碘溶液中。

代表数据

  1. 可以预期淀粉合酶活性结果的代表性实例如图2(b)(来自图8A,Li等人,2011)和小麦籽粒中的图2(c)图2,McMaugh等人,2014)。


    图2.大麦和小麦发育的胚乳的淀粉合酶活性(a)。用碘溶液染色前的天然PAGE凝胶。 (b)。显示在用碘溶液染色后在大麦发育胚乳中的淀粉合酶I和IIIa活性的天然PAGE凝胶。 (C)。显示在用碘溶液染色后小麦发育胚乳中的淀粉合酶I和IIIa活性的天然PAGE凝胶。
  2. 这个协议在我们手中是高度可重现的。天然聚丙烯酰胺凝胶的质量决定了蛋白质条带的质量。 TEMED和APS的质量可能影响凝胶质量,然后影响蛋白质带质量

笔记

  1. 来自牡蛎的糖原是天然聚丙烯酰胺凝胶(天然PAGE)的最佳底物。

食谱

  1. 提取缓冲
    加入20μl1M磷酸钾缓冲液,pH7.5(终浓度:50mM KPi) 加入1μl500mM EDTA,pH7.5(终浓度:5mM EDTA)
    加入400μl50%甘油(最终浓度:20%甘油) 加入567μlMilliQ H sub 2 O 2 / 添加蛋白酶抑制剂混合物为植物细胞和组织提取物和DTT缓冲液,如下准备提取
    添加2微升蛋白酶抑制剂鸡尾酒植物细胞和组织提取物
    加入10μl500mM DTT(终浓度:5mM DTT)
  2. 1×电泳缓冲液
    加入80 ml 10x运行缓冲液
    添加0.15 g/L的DTT
    用蒸馏水补足至800毫升 10x运行缓冲液(10x Tris-甘氨酸储备液)
    1.9M甘氨酸(144g/L) 250mM Tris(30.3g/L)

致谢

该协议从以前的出版物(Abel等人,1996)修改。作者感谢Behjat Kosar-Hashemi,Emma Anschaw,Steve McMaugh,Sapna Vibhakaran Pillai和Hong Wang对这个协议的优化和测试的贡献。作者还感谢CSIRO植物工业,CSIRO食品未来旗舰和ACVL有限公司在研究过程中提供资金资源测试本协议。

参考文献

  1. Abel,G.J.,Springer,F.,Willmitzer,L。和Kossmann,J。(1996)。 对编码来自马铃薯的新型139kDa淀粉合酶的cDNA的克隆和功能分析( Solanum tuberosum L.)。植物J 10(6):981-991。
  2. Li,Z.,Mouille,G.,Kosar-Hashemi,B.,Rahman,S.,Clarke,B.,Gale,K.R.,Appels,R.and Morell,M.K。(2000)。 小麦淀粉合成酶III基因的结构和表达。表达基因中的基序定义淀粉合酶III基因家族的谱系。植物生理学123(2):613-624。
  3. Li,Z.,Li,D.,Du,X.,Wang,H.,Larroque,O.,Jenkins,C.L.,Jobling,S.A.and Morell,M.K。 大麦amo1基因座与淀粉合成酶IIIa基因紧密连锁,并负调节颗粒结合的表达淀粉合成基因。 J Exp Bot 62(14):5217-5231。
  4. McMaugh,SJ,Thistleton,JL,Anschaw,E.,Luo,J.,Konik-Rose,C.,Wang,H.,Huang,M.,Larroque,O.,Regina,A.,Jobling, ,MK和Li,Z.(2014)。 淀粉合成酶I表达的抑制影响小麦胚乳中淀粉的颗粒形态和颗粒大小和精细结构。 65(8):2189-2201。
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引用:Li, Z. (2016). Analysis of Starch Synthase Activities in Wheat Grains using Native-PAGE. Bio-protocol 6(2): e1713. DOI: 10.21769/BioProtoc.1713.
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