Malat-dehydrogenase

Tomato

MDH – (Malat-dehydrogenase)

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 pH 3-10 (Cat.No. 42008) – 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.

Cathode fluid 10
Bring 0.90 g L-arginine base (Sigma A- 5006)+ 0.70 g L-lysine (Sigma L-5501)+ 24.0 ml ethylene diamine (q.c.w. Sigma E-9890) in 200 ml water.  Store in refrigerator.

Anode fluid 3
Bring 0.68 g L-glutamic acid (q.c.w. Sigma G-6904) and 0.72 L-aspartic acid (q.c.w. Sigma A-8949) in 200 ml distilled water. Stir until fully dissolved. 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.

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

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

0.1 M Tris-HCl pH 8.5
Bring 24.2 g Tris (q.c.w. Sigma T-8524) and 1600 ml distilled water in a beaker. Stir until fully dissolved. Adjust the pH to 8.5 with 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.

MDH extraction buffer 0.025 M Tris pH 8.5
6.0 ml 0.1 M Tris-HCl pH 8.5 + 18.0 ml distilled water. Add 250 µl (± 10 drops out of a plastic dropper bottle) Triton X-100  (q.c.w. Sigma X-100) and 0.05 g dithiothreitol (DTT) (q.c.w. Sigma D – 9163).  Mix well before use! Add a trace (tip of a small spatula) of Orange G (q.c.w.  Sigma O – 1625).  Do not store.

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

2 M DL – malic acid
Bring in beaker 80.0-g sodium hydroxide pellets (q.c.w Sigma S-8045). Add slowly 400 ml distilled water to the pellets. The solution is getting hot!  Let the solution cool down and add little by little 134.0 g DL-malic acid (q.c.w. Sigma M – 0875). Mix well. Adjust the pH to 7.0 with 4 N sodium hydroxide (approx. 6 ml) Bring to a final volume of 500 ml with distilled water. Store in refrigerator

2 % v/v Acetic acid solution
Bring 20 ml of acetic acid 100% (q.c.w. BDH 27013) in 2000 ml distilled water. Store at room temperature

Gel preparation
9.5 ml        16 % glycerol
2.0 ml        Acrylamide/bis acrylamide 29: 1 solution
1.0 ml        pH 3-10 (ProTec Bioseparation Cat. No.42008)
0.065 ml    0.1 % w/v riboflavin
0.012 ml    TEMED
0.035 ml    10 % w/v ammonium persulfate

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

Sample preparation
Single seeds are placed into a 96 well micro plate. To each well, 100 µl of MDH extraction solution is added. The seeds are homogenized and centrifuged for 10 minutes at 3000 rpm. at 10 °C (or at room temperature).

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 four parts. Space the electrodes evenly across the gel, alternating cathode (black electrode) and anode (red electrode). Place the electrode plateau directly onto the gel, making electrode imprints in the gel. Blotting paper wicks (1 x 6-x 260 mm) are used. Wet the cathode wicks with cathode fluid 10 and the anode wicks with anode fluid 3. Gently blot the wicks but keep them fairly wet. Place the cathode wicks onto the cathode imprints in the gel and the anode wicks onto the anode imprint. Bring the electrode plateau on top of the electrode wicks.

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

Prefocusing
Run 1: 600 Volts – 60 mA – 12 Watts – 75 Volthours

Sample application
After the prefocusing step, the 96 templates are positioned ± 5 mm from the anodal wick. See cartoon image section “gel interpretation.
Each sample well is filled with 15 ul of supernatant.

Focusing
Run 2:   200 Volts – 60 mA – 12 Watts –   50 Volthours
Run 3: 1000 Volts – 60 mA – 12 Watts – 700 Volthours

MDH staining
200 ml       0.1 M Tris- HCl pH 8.5
  0.3 ml      4 M MgCl2 x 6 H2O
10.0 ml      2 M DL-malic acid
0.100 g    NAD (q.c.w. Sigma N-7004)
0.050 g    MTT (q.c.w. Sigma M-2128)
0.010 g    PMS (q.c.w. Sigma P-9625) (tip of a small spatula)

Heat up the Tris-HCl buffer solution to ± 37 °C. Add the reagents together and mix until fully dissolved and stain the gel at ± 37°C or room temperature until the bands can be clearly visualized. Remove the stain 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)