Materials and Reagents

Mature corn kernels
Petroleum ether
Ethanol
β-mercaptoethanol
Sodium dodecyl sulfonate (SDS)
Urea
Glycerol
HCl
Tris(Hydroxymethyl)aminomethane (Tris)
Phenylmethanesulfonyl fluoride (PMSF)
Methylene diacrylamide
Bromophenol blue
CHAPS (Sigma-Aldrich, catalog number: V900480-5G )
Dithiothreitol (DTT)
Liquid nitrogen
ddH₂O
SDS-PAGE gel (15% separation)
Coomassie brilliant blue (R250) staining buffer
30% acrylamide (see Recipes)
Sodium borate buffer (see Recipes)
5x protein loading buffer (see Recipes)
IPG solution (see Recipes)

Equipment

A mortar and pestle
Centrifuge (Eppendorf, model: 5415D )
Shaker (Zhicheng, model: ZHWY-111C )
Concentrator plus (Eppendorf, catalog number: 5305000.193 )
Gel Doc^TM XR + System (Bio-Rad, catalog number: 170-8195 )

Procedure

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.
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

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

References

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.
Gibbon, B. Protein extraction from flour [updated April 2003]. Available from http://ag.arizona.edu/research/larkinslab/protocols.htm
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.
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.
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.