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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, 2820 views, 0 Q&A, How to cite
DOI: http://dx.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)

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.


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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