Quality traits. Gluten Strength

Methods. Protein Extraction

Contributed by Laura Pfluger (lpfluger@cirn.inta.gov.ar)

Protein extraction from seeds starts with a grinding step followed by a centrifugation step that are both common for glutenin and gliadin extraction.

1. Proteins are extracted from single seed by grinding them in a mortar with 1.5M dimethyl formamide (DMF). The solvent volume to seed-weight ratio is 5:1 (µl : mg).
2. The solution is centrifuged for 10 min at 14000 xg and the supernatant is stored at -20°C for gliadin analysis.
3. The pellet is treated with 1 ml 0.08 M Tris-HCl buffer (pH 8.5) containing 1% SDS for 30 min with intermittent vortexing.
4. Centrifugation for 8 min at 14000 xg.
5. Repeat the extraction (steps 3 and 4) and discard the supernatant.
6. Resuspend the pellet with 0.08M Tris-HCl buffer (pH 8.5) containing dithiotreitol (DTT), (1.5% w/v) and SDS (1% w/v).
7. Extract for 30 min at 60°C with occasional vortexing followed by centrifugation (10 min at 14000 xg).
8. Take 200 µl of each supernatant for alkylation with 2.8 µl of 4-vinyl-pyridine.
9. Incubate at 60°C for 30 min with two vortexings.
10. Add acetone (1 ml) at each sample to precipitate glutenins.
11. Leave samples overnight at -20°C.
12. Centrifuge (10 min a 14000 xg), discarding the supernatant.
13. Dissolve pellets with 0.25 M Tris-HCl buffer (pH 6.8) containing 1.5% DTT. The solvent volume to seed weight ratio is 5:1 (µl : mg) and proceed with glutenin analysis