Maize Endosperm Protein Extraction and Analysis

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 Doc^TM XR + System (Bio-Rad, catalog number: 170-8195 )
   

Procedure

1. 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.
       
       
2. 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.