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Alcohol-solubility is the most characteristic feature of the zein proteins, the major storage protein in maize. Using sodium borate buffer system with added reducing agent, total proteins are isolated, and zein proteins are separated from non-zein proteins. The extraction effect is intuitive on a SDS-PAGE isolation system. In addition, a simple and rapid approach to extract zeins is introduced, taking full advantage of alcohol-solubility of zeins directly.

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Maize Endosperm Protein Extraction and Analysis
玉米胚乳蛋白的提取和分析

植物科学 > 植物生物化学 > 蛋白质 > 分离和纯化
作者: Xinze Chen
Xinze ChenAffiliation: School of Life Sciences, Shanghai University, Shanghai, China
Bio-protocol author page: a704
Dongsheng Yao
Dongsheng YaoAffiliation: School of Life Sciences, Shanghai University, Shanghai, China
Bio-protocol author page: a705
 and Rentao Song
Rentao SongAffiliation: School of Life Sciences, Shanghai University, Shanghai, China
For correspondence: rentaosong@staff.shu.edu.cn
Bio-protocol author page: a706
Vol 3, Iss 14, 7/20/2013, 3686 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.832

[Abstract] Alcohol-solubility is the most characteristic feature of the zein proteins, the major storage protein in maize. Using sodium borate buffer system with added reducing agent, total proteins are isolated, and zein proteins are separated from non-zein proteins. The extraction effect is intuitive on a SDS-PAGE isolation system. In addition, a simple and rapid approach to extract zeins is introduced, taking full advantage of alcohol-solubility of zeins directly.
Keywords: Maize(玉米), Seed(种子), Zein(玉米醇溶蛋白), Extraction(萃取)

[Abstract]

Materials and Reagents

  1. Mature corn kernels
  2. Petroleum ether
  3. Ethanol
  4. β-mercaptoethanol
  5. Sodium dodecyl sulfonate (SDS)
  6. Urea
  7. Glycerol
  8. HCl
  9. Tris(Hydroxymethyl)aminomethane (Tris)
  10. Phenylmethanesulfonyl fluoride (PMSF)
  11. Methylene diacrylamide
  12. Bromophenol blue
  13. CHAPS (Sigma-Aldrich, catalog number: V900480-5G )
  14. Dithiothreitol (DTT)
  15. Liquid nitrogen
  16. ddH2O
  17. SDS-PAGE gel (15% separation)
  18. Coomassie brilliant blue (R250) staining buffer
  19. 30% acrylamide (see Recipes)
  20. Sodium borate buffer (see Recipes)
  21. 5x protein loading buffer (see Recipes)
  22. IPG solution (see Recipes)

Equipment

  1. A mortar and pestle
  2. Centrifuge (Eppendorf, model: 5415D )
  3. Shaker (Zhicheng, model: ZHWY-111C )
  4. Concentrator plus (Eppendorf, catalog number: 5305000.193 )
  5. Gel DocTM XR + System (Bio-Rad, catalog number: 170-8195 )

Procedure


I.   Using sodium borate buffer system to extract zeins

  1. Soak 3-5 mature corn kernels in ddH2O for 10 min, then remove the pericarp and embryo and dry the kernels for 10 min at 37 °C.
  2. Grind kernels into powder using a mortar and pestle within liquid nitrogen.
  3. Transfer the powder into a 2 ml eppendorf tube. Dry it in Concentrator plus for 1 h till achieve constant weight.
  4. Add 1 ml petroleum ether. Vortex and place in the shaker at 250 rpm for 1 h.
  5. Centrifuge for 15 min at 12,000 rpm at room temperature (RT), and discard the supernatant.
  6. Dry it in Concentrator Plus for 1.5 h until no smell of organic liquid is detectable.
  7. Fill a new 2 ml Eppendorf tube with 50 mg dried powder from the step 6.
  8. Add 1 ml sodium borate buffer and 20 μl β-mercaptoethanol as well as 1% PMSF. Mix and incubate with shaking at 250 rpm for at least 2 h at 37 °C.
  9. Centrifuge for 15 min at 12,000 rpm at RT.
  10. Transfer 300 μl supernatant into a new 2 ml eppendorf tube as total protein extraction (Fraction A).
  11. Transfer another 300 μl supernatant from the step 9 into a new 2 ml Eppendorf tube, and add 700 μl ethanol as well as 1% PMSF. Mix with shaking at 250 rpm for 2 h at RT.
  12. Centrifuge product from step 11 for 15 min at 12,000 rpm at RT.
  13. Transfer 400-500 μl supernatant into a new 2 ml Eppendorf tube and dry it in Concentrator plus for 2-3 h. Resuspend it in 200 μl IPG as zein proteins extraction (Fraction B).
  14. Wash the precipitate in step 12 with 70% ethanol twice.
  15. Centrifuge for 15 min at 12,000 rpm at RT.
  16. Discard the supernatant and air-dry the precipitate until the edges become transparent. Resuspend it in 200 μl IPG as non-zein proteins extraction (Fraction C).
  17. Add 10 μl protein extraction from fraction A, or B, or C (see the attached corresponding pictures), 1.5 μl DTT and 3 μl 5x Protein loading buffer in a new 0.2 ml eppendorf tube. Heat 10 min at 99 °C for denaturation.
  18. Load 2-5 μl denatured protein sample and perform the SDS-PAGE on a 15% separation gel.
  19. Afterwards, the gel is stained with Coomassie brilliant blue R250 (Figure 1, Figure 2, and Figure 3).


    Figure 1. SDS-PAGE (Fraction A). M represents protein standards with molecular weight ranging from 14,400 to 97,400 Da (similarly hereinafter). This figure shows total proteins extraction. 1, 2, 3, 4 represent four different maize cultivars.


    Figure 2. SDS-PAGE (Fraction B). This figure shows zein protein extraction. 1, 2 represent two different maize cultivars (two replicates for each maize cultivar).


    Figure 3. SDS-PAGE (Fraction C). This figure shows non-zein protein extraction. 1, 2, 3, 4 represent four different maize cultivars.


II.  Simple and rapid extraction approach

  1. Soak 3-5 mature corn kernels in ddH2O for 10 min, then remove the pericarp and embryo and dry the kernels for 10 min at 37 °C.
  2. Grind kernels into powder using a mortar and pestle within liquid nitrogen.
  3. Transfer the powder into a 2 ml eppendorf tube. Dry it in Concentrator plus for 1 h till achieve constant weight.
  4. Fill a new 2 ml eppendorf tube with 50 mg dry powder from step 3.
  5. Add 400 μl 70% ethanol and 8 μl β-mercaptoethanol as well as 1% PMSF.
  6. Mix and incubate for at least 2 h at RT. Invert the cube 2-3 times during incubation.
  7. Centrifuge for 10 min at 1,300 rpm at RT.
  8. Transfer 100 μl supernatant into a new 2 ml eppendorf tube.
  9. Add 10 μl 10% SDS, and mix by pipetting.
  10. Dry it in Concentrator plus for 1 h.
  11. Add 200 μl ddH2O for elution.
  12. SDS-PAGE is performed in 15% polyacrylamide gels, and the gels are stained with Coomassie brilliant blue R250 (Figure 4).


    Figure 4. SDS-PAGE (zein proteins). This figure shows zein proteins through simple and rapid extraction approach. 1, 2 represent two replicates of same maize cultivar.

Recipes

  1. 30% acrylamide (500 ml aqueous solution)
    145 g acrylamide
    5 g methylene diacrylamide
  2. Sodium borate buffer
    12.5 mmol/L sodium borate
    1% SDS
    pH 10.0
  3. 5x Protein Loading Buffer
    60 mmol/L Tris-HCl (pH 6.8)
    25% glycerol
    2% SDS
    0.1% bromophenol blue
  4. IPG solution
    8 mol/L urea
    2% CHAPS

References

  1. Bradford, M. M. (1976). A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein–dye binding. Anal Biochem 72: 248-254.
  2. Gibbon, B. Protein extraction from flour [updated April 2003]. Available from http://ag.arizona.edu/research/larkinslab/protocols.htm
  3. Wallace, J. C., Lopes, M. A., Paiva, E. and Larkins, B. A. (1990). New Methods for Extraction and Quantitation of Zeins Reveal a High Content of gamma-Zein in Modified opaque-2 Maize. Plant Physiol 92(1): 191-196.
  4. Wang, G., Sun, X., Wang, G., Wang, F., Gao, Q., Sun, X., Tang, Y., Chang, C., Lai, J., Zhu, L., Xu, Z. and Song, R. (2011). Opaque7 encodes an acyl-activating enzyme-like protein that affects storage protein synthesis in maize endosperm. Genetics 189(4): 1281-1295.
  5. Wu, Y., Goettel, W. and Messing, J. (2009). Non-Mendelian regulation and allelic variation of methionine-rich delta-zein genes in maize. Theor Appl Genet 119(4): 721-731.

材料和试剂

  1. 成熟玉米粒
  2. 石油醚
  3. 乙醇
  4. β-巯基乙醇
  5. 十二烷基磺酸钠(SDS)
  6. 尿素
  7. 甘油
  8. HCl
  9. 三(羟甲基)氨基甲烷(Tris)
  10. 苯基甲磺酰氟(PMSF)
  11. 亚甲基二丙烯酰胺
  12. 溴酚蓝
  13. CHAPS(Sigma-Aldrich,目录号:V900480-5G)
  14. 二硫苏糖醇(DTT)
  15. 液氮
  16. ddH sub 2 O
  17. SDS-PAGE凝胶(15%分离)
  18. 考马斯亮蓝(R250)染色缓冲液
  19. 30%丙烯酰胺(见配方)
  20. 硼酸钠缓冲液(见配方)
  21. 5x蛋白质上样缓冲液(见配方)
  22. IPG解决方案(参见配方)

设备

  1. 研钵和杵
  2. 离心机(Eppendorf,型号:5415D)
  3. 摇床(Zhicheng,型号:ZHWY-111C)
  4. Concentrator plus(Eppendorf,目录号:5305000.193)
  5. Gel Doc TM XR +系统(Bio-Rad,目录号:170-8195)

程序


I.   使用硼酸钠缓冲系统提取玉米醇溶蛋白

  1. 在ddH 2 O中浸泡3-5个成熟玉米粒10分钟,然后取出果皮和胚,并在37℃下干燥谷粒10分钟。
  2. 使用研钵和杵在液氮中将谷粒研磨成粉末
  3. 将粉末转移到2毫升eppendorf管中。 在浓缩器中加干燥1小时,直至达到恒重
  4. 加入1ml石油醚。 涡旋,并在振荡器中以250rpm放置1小时
  5. 在室温(RT)下以12,000rpm离心15分钟,弃去上清液
  6. 在Concentrator Plus中干燥1.5小时,直到没有可检测到有机液体的气味
  7. 用50毫升来自步骤6的干燥粉末填充新的2毫升Eppendorf管
  8. 加入1ml硼酸钠缓冲液和20μlβ-巯基乙醇以及1%PMSF。 混合并在37℃下以250rpm振荡孵育至少2小时
  9. 在室温下以12,000rpm离心15分钟
  10. 转移300微升上清液到一个新的2毫升eppendorf管作为总蛋白提取(分数A)
  11. 转移从步骤9另一个300微升上清液到一个新的2毫升Eppendorf管,并添加700微升乙醇以及1%PMSF。 在室温下以250rpm振摇混合2小时
  12. 将步骤11的产物在室温下以12,000rpm离心15分钟
  13. 将400-500μl上清液转移到新的2ml Eppendorf管中,并在Concentrator plus中干燥2-3小时。 重悬在200微升IPG作为玉米醇溶蛋白提取(部分B)
  14. 在步骤12中用70%乙醇洗涤沉淀两次
  15. 在室温下以12,000rpm离心15分钟
  16. 弃去上清液并风干沉淀,直到边缘变得透明。 重悬在200微升IPG作为非玉米蛋白提取(分数C)
  17. 加入10μl蛋白提取从A部分,或B或C(见附图相应的图片),1.5微升DTT和3微升5x蛋白加载缓冲液在新的0.2毫升eppendorf管中。 在99℃下加热10分钟以使其变性
  18. 加载2-5μl变性蛋白质样品,并在15%分离凝胶上进行SDS-PAGE
  19. 然后,凝胶用考马斯亮蓝R250染色(图1,图2和图3)。


    图1. SDS-PAGE(级分A)。 M代表分子量范围为14,400至97,400 Da(以下相似)的蛋白标准品。该图显示了总蛋白提取。 1,2,3,4代表四个不同的玉米品种

    图2. SDS-PAGE(组分B)。此图显示 玉米醇溶蛋白提取。 1,2代表两种不同的玉米品种 (每个玉米品种两次重复)

    图3.DSC-PAGE(级分C)。 此图显示非玉米醇溶蛋白提取。 1,2,3,4代表四种不同的玉米品种。


II。  简单快速提取方法

  1. 在ddH 2 O中浸泡3-5个成熟玉米粒10分钟,然后取出果皮和胚,并在37℃下干燥谷粒10分钟。
  2. 使用研钵和杵在液氮中将谷粒研磨成粉末
  3. 将粉末转移到2毫升eppendorf管中。 在浓缩器中加干燥1小时,直至达到恒重
  4. 用50mg来自步骤3的干粉填充新的2ml eppendorf管
  5. 加入400μl70%乙醇和8μlβ-巯基乙醇以及1%PMSF
  6. 混合并在室温下孵育至少2小时。 在孵育期间将立方体颠倒2-3次
  7. 在室温下以1,300rpm离心10分钟
  8. 将100μl上清液转移到新的2ml eppendorf管中
  9. 加入10μl10%SDS,并通过移液混合
  10. 在浓缩器中干燥1小时。
  11. 加入200μlddH 2 O用于洗脱
  12. SDS-PAGE在15%聚丙烯酰胺凝胶中进行,凝胶用考马斯亮蓝R250染色(图4)。


    图4. SDS-PAGE(玉米醇溶蛋白)。该图显示了玉米醇溶蛋白 通过简单快速的提取方法。 1,2表示两个 相同玉米品种的重复

食谱

  1. 30%丙烯酰胺(500ml水溶液) 145克丙烯酰胺 5克亚甲基二丙烯酰胺
  2. 硼酸钠缓冲液
    12.5 mmol/L硼酸钠 1%SDS
    pH 10.0
  3. 5x蛋白质上样缓冲液
    60 mmol/L Tris-HCl(pH 6.8)
    25%甘油 2%SDS
    0.1%溴酚蓝
  4. IPG解决方案
    8 mol/L尿素
    2%CHAPS

参考文献

  1. Bradford,M.M。(1976)。 使用蛋白质 - 染料结合原理的微克数量蛋白质定量的快速灵敏的方法。 Anal Biochem 72:248-254。
  2. Gibbon,B.蛋白质提取从面粉[2003年4月更新]。 可从http://ag.arizona.edu/research/larkinslab/protocols.htm? a>
  3. Wallace,J.C.,Lopes,M.A.,Paiva,E。和Larkins,B.A。(1990)。 新的提取和定量玉米蛋白的新方法揭示了修饰玉米蛋白的高含量opaque -2 Maize。 Plant Physiol 92(1):191-196。
  4. Wang,G.,Sun,X.,Wang,G.,Wang,F.,Gao,Q.,Sun,X.,Tang,Y.,Chang,C., Xu,Z.和Song,R。(2011)。 Opaque7编码酰基 - 激活酶样蛋白质,其影响玉米胚乳中的储存蛋白质合成。 Genetics 189(4):1281-1295。
  5. Wu,Y.,Goettel,W。和Messing,J。(2009)。 非孟德尔调节和玉米中富含甲硫氨酸的δ-玉米醇溶蛋白基因的等位基因变异。 T 119(4):721-731。
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How to cite this protocol: Chen, X., Yao, D. and Song, R. (2013). Maize Endosperm Protein Extraction and Analysis. Bio-protocol 3(14): e832. DOI: 10.21769/BioProtoc.832; Full Text



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