Squash

ME – (Malic enzyme)

16 % v/v glycerol
Bring 200 grams (= 160 ml) of glycerol (q.c.w. Sigma G-7757) and 800 ml distilled water in a beaker. Mix well and bring to a final volume of 1000 ml with distilled water. Store in the refrigerator.

Sepalyte Squash (Cat. No. 42100 ProTec Bioseparation)
Ready to hand Sepalyte solutions. Store in refrigerator

Acrylamide/ bis acrylamide 29 : 1 solution
Ready to hand acrylamide solution (q.c.w. Sigma A-3574). Store in refrigerator.

0.1N Sodium hydroxide
4.0 g sodium hydroxide (q.c.w. Sigma S-8045) in 1000 ml distilled water. Add a trace (tip of a small spatula) of sodium azide (q.c.w. Sigma S-8032). Store at room temperature

0,1% w/v riboflavin
0.05 g riboflavin (q.c.w. Sigma R-0508) in 50 ml 0.1 N sodium hydroxide.
Store in the refrigerator for up to 2 weeks.

10% w/v ammonium persulfate
0.5 g ammonium persulfate (q.c.w. Sigma A- 3678) in 5.0 ml distilled water.
Store in refrigerator for one week

4 N Sodium hydroxide
16.0 g sodium hydroxide (q.c.w. Sigma S-8045) in 100 ml distilled water. Store in the refrigerator.

ME extraction solution
50 mg dithiothreitol (q.c.w. Sigma D-9163),10 g sucrose (q.c.w. Sigma S-8501) in 100 ml distilled water. Mix well before use! Add a trace (tip of a small spatula) of Orange G (q.c.w. Sigma O-1625). Do not store.

2 M DL-Malic acid
Dissolve 27.0 g DL-malic acid (q.c.w. Sigma M-0875) in approximately 40 ml DI water. Mix well. Adjust the pH to 7.0 with sodium hydroxide. Bring to a final volume of 100 ml with distilled water. Store in refrigerator.

0.1 M Tris-HCl pH 8.0
Bring 24.2 g Tris (q.c.w. Sigma T-8524) and 0.8 g EDTA (q.c.w. Sigma E-5134) in 1600 ml distilled water. Stir until fully dissolved. Adjust the pH to 8.0 with concentrated HCl (q.c.w. BDH 28507). Bring to a final volume of 2000 ml with distilled water. Add a trace (tip of a small spatula) of sodium azide (q.c.w. Sigma S-8032). Store at room temperature.

4 M magnesium chloride
Bring 203-g magnesium chloride hexahydrate (q.c.w. Sigma M-0250) and 150 ml distilled water in a beaker. Stir until fully dissolved and bring to a final volume of 250 ml. Store at room temperature.

2 % v/v Acetic acid solution
Bring 20 ml of acetic acid 100%(q.c.w. BDH 27013) in 1000 ml distilled water. Store at room temperature.

TEMED
Ready to use TEMED Sigma ( T-9281).

Gel preparation
9.5 ml        16 % glycerol
2.0 ml        Acrylamide/bis 29:1solution
2.0 ml        Sepalyte Squash
0.065 ml    0.1 % w/v riboflavin
0.012 ml    TEMED
0.035 ml    10% ammonium persulfate

Add the above reagents and swirl to mix. Pour the gel according to the flap technique and allow polymerizing for at least 4 hours under a light. Store the gels in a sealed bag in the refrigerator for up to 2 weeks.

Sample preparation
By means of a “hole puncher diameter 5 mm” some endosperm is punched out, split in two and one half is put in a 96 microtiter plate. Crush the seeds using the ISOLAB crusher and rubber hammer. To each well 190 ml ME extraction solution is added, homogenized for 3 minutes, using the Terminator, and centrifuged for 10 min. at 3000 rpm. at 10° C (or at room temperature). Transfer clear
supernatant (about 80 µl to a clean 96 well plate and centrifuge for 10 minutes at 3000 rpm at 10º C (or at room temperature). Repeat centrifugation if necessary.

Electrophoresis
Turn the cooling supply on and set at a temperature of ± 15°C. Remove the gel from the glass plates. Clean the back of the gel with methanol/ethanol. Place the gel onto the cooling plate with several ml of water. The gel can be divided into three parts. Space the electrodes evenly across the gel, alternating cathode (black electrode) and anode (red electrode) and then place directly onto the gel.

Power settings are for one gel (double the mA and Watts when running two gels).

Prefocusing
Run1:150V–50mA–30W–20min.
Run1:150V-50mA-15W-20min (3parts)

Sample application
After the prefocusing step, the 52 templates are positioned ± 20 mm from the cathode (black electrode). See cartoon image section “gel interpretation 9.1.6 “ Each sample well is filled with 15 µl of supernatant.

Focusing
Run 2: 300 V–50 mA–30 W–10 min.
Run 3: 450 V–50 mA–30 W–10 min.
Run 4: 600 V–50 mA–30 W–10 min.
Run 5: 900 V–50 mA–30 W–10 min.
Run 6: 1000 V–50 mA–30 W–50 min.
(gel in three parts, stop focusing)
Run 7: 1200 V–50 mA–30 W–10 min.
Run 8: 1350 V–50 mA–30 W–10 min.
Run 9: 1500 V–50 mA–30 W–20 min.

After the gel has finished running, remove the gel from the cooling plate and place into an appropriate staining tray.

ME staining
200 ml 0.1 M Tris- HCl pH 8.0
0.32 ml 4.0 M MgCl₂ solution
10 ml 2M DL-malic acid
0.05 g NADP (Sigma N-3886)
0.05 g MTT (Sigma M-2128)
0.01 g PMS (Sigma P-9625) (tip of a small spatula).

Heat up the Tris-HCl solution to ± 37° C. Add the reagents together and mix until fully dissolved and stain the gel at ± 37° C or at room temperature until the bands can be clearly visualized. Remove the staining solution and destain the gel in a 2 % glacial acetic acid solution for about 10 minutes and rinse with distilled water for an additional 10 minutes. The gel can then be air-dried.

Gel interpretation
No international enzyme classification is used.
The nearest band of interest from the anode is genotyped “11”
The farthest band of interest from the anode is genotyped “ 22”
The hybrid is genotyped “1/2” in case that the female is 11 and the male 22.
The hybrid is genotyped “2/1” in case that the female is 22 and the male 11.

ProTec Bioseparation (Protein Electrophoresis)