Quality traits. Gluten Strength
Methods. SDS-PAGE
High molecular weight glutenin subunits are analysed by Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE) in 8% or 10% polyacrylamide gel (T=8% or T=10%) with 1.28% cross-linker concentration (C=1.28%). Electrophoretic separations are carried out at 30 mA/gel. For details on seed protein extraction follow this link.
REAGENTS:
- Acrylamide solution (40% w/v)
- N,N' methylene-bisacrylamide solution (2% w/v)
- Separating buffer: Tris-HCl 3M, pH 8.8
- 91g Tris
- HCl to pH 8.8
- Distilled water up to 250 ml
- Stacking buffer: Tris-HCl 0.5M pH 6.8:
- 6g Tris
- HCl to pH 6.8
- Distilled water up to 100 ml
- Electrode buffer (10x):
- 30.3 g Tris
- 144 g glycin
- 10 g SDS
- Distilled water up to 1000 ml
- Ammonium persulphate (APS) solution (10%)
- SDS solution (10%)
- N,N,N',N' tetramethyl-ethylen-diamine (TEMED)
Stacking gel (for 2 gels of 1mm thickness), C=2.67%, T=3.7%:
- 0.912 ml acrylamide solution (40%)
- 0.5 ml bis-acrylamide solution (2%)
- 2.5 ml stacking buffer
- 6.0 ml distilled water
- Degas and add:
- SDS (10%) 100 µl
- APS (10%) 75 µl
- TEMED 13 µl
Separating gel (for 2 gels of 1mm thickness) C=1.28%; T= 8% , T=10% and T=12%.
T values of 8% and 10% are recommended for separating HMW glutenins and gels with T=12% for improved separation of LMW glutenins
|
T=8% |
T=10% |
T=12% |
| Acrylamide solution (40%) |
10 ml |
12.34 ml |
14.80 ml |
| Bis-acrylamide solution (2%) |
2.56 ml |
3.2 ml |
3.84 ml |
| Separating buffer |
6.50 ml |
6.50 ml |
6.50 ml |
| Distilled water |
30.28 ml |
27.30 ml |
24.20 ml |
Degas and add:
- 500 µl SDS (10%)
- 120 µl APS (10%)
- 36-44µl TEMED
STAINING
The staining procedure is the same for both, SDS-PAGE and A-PAGE gels.
Gels are stained with 0.2% (w/v) Coomassie Blue R-250, in 5% (v/v) ethanol and 12% (w/v) trichloroacetic acid overnight and destained in tap water for 24h.
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| Allelic variants for HMW-Gs in bread and durum wheat. |
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| Some combination of HMW-Gs alleles in eleven wheat varieties |
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