Marker Assisted Selection in Wheat Marker Assisted Selection in Wheat - HOMECSREES-USDA

Quality traits. Gluten Strength

Methods. A-PAGE

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

This is the electrophoretic method of choice for the characterization of gliadins. The main difference with PAGE electrophoresis is the use of a aluminum lactate / lactic acid buffer. Follow this link for details on protein extraction for analyzing gliadins and glutenins.

REAGENTS

Acrylamide solution:

  • 28.57 g acrylamide
  • 1.43 g bis-acrylamide

Make up to a final volume of 100 ml.

Stock buffer:

  • 31.25 g aluminum lactate
  • 50 ml lactic acid

Make up to a final volume of 500 ml.

Gel preparation (for one gel)

Glass handling tip: Clean them very carefully and handle them with gloves!!!!

Solution 1 (prepare 1st the following ):

  • 40 µl ascorbic acid (100 mg in 400 µl, final volume)
  • 4 µl SO4Fe (40 mg in 1 ml, final volume)
  • 45 µl H2O2 (300 µl in 3.5 ml of final volume)

Note: Prepare the gels one by one because the polymerization is very quick.

Solution 2:

  • 8.4 ml acrylamide stock solution.
  • 0.5 ml stock buffer
  • 30.7 ml H2O

Mix quickly solutions 1 and 2 and load the gel with a syringe. Load quickly at the beginning and slowly once the solution reaches the comb.

After 15-20 minutes take out the comb and rinse the wells two times with running buffer.

Running buffer: Take 100 ml of stock buffer and make it up to a final volume of 5 l.

The buffer on the bottom chamber may be used twice: save it in the refrigerator and use it within one week.

RUNNING THE GEL

Invert the electrodes (black-red; red-black) both for pre running and running!!!!

1) Pre running:

  • Pre run the gel for 1 1/2 h at 25mA / gel.
  • Ideal temperature : 12°C

2) Running:

  • Load the gel (5 µl of sample / well, if using combs of 15 teeth). In the first well load 2 µl of metyl violet as running marker. The running finishes when the marker goes completely out of the gel)
  • Run the gel at 25 mA / gel. Ideal temperature: 15 °C

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.


A-PAGE. Differences in gliadin patterns for five
wheat lines