PGM

Squash

PGM – (Phosphoglucomutase)

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

Sepalyte pH 3-6 (Cat. No.42006 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
Dissolve 0.5 grams in 5 ml dH2O. Store in refrigerator.

0.5 % v/v  (3-10 ) Sepalyte solution
2.5 ml Sepalyte (3- 10) in 500 ml distilled water. Add a trace (tip of a small spatula) of Orange G (q.c.w. Sigma O-1625). Store in refrigerator

0.2 M Tris-HCl pH 8.0
Bring 48.4 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 (± 24 ml) 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.

Glucose-6-phosphate-dehydrogenase (Sigma G 7750)
Dissolve 2000 U in 10 ml water and divide in 0.4 ml in eppendorf vials, equal to  80 U per vial. Store the vials in the freezer.

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.

Gel preparation
9.5 ml        16 % glycerol
2.0 ml        Acrylamide/bis acrylamide 29: 1 solution
0.7 ml        Sepalytes (pH 3-6 (Cat. No.42006)
0.3 ml        Sepalytes (3-10)
0.065 ml     0.1 % w/v riboflavin
0.035 ml     10 % w/v Ammonium persulfat
0.012 ml     TEMED

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
Single cotyledon/first leaf punches (using the cork borer # 3, diameter 7 mm) from soil germinated seeds (6-8 days old) are submerged in water. The cotyledons absorb water. Place the wet cotyledons into a 96 well microplate. Flatten the punch-outs onto the bottom of the well. Freeze for at least 2 hours or better overnight. The punch-outs should be homogenized while they are still frozen. Add now to each well 150 ml of 0.5 % Sepalyte 3-10 (cold). Crush for 3 minutes with the Terminator and centrifuge for 10 minutes at 3000 rpm. at 10° C  (or at room temperature). Transfer 75 μl of clear supernatant to a clean 96 well microplate and centrifuge for 10 minutes at 3000 rpm at 10° C.

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) and then place directly onto the gel.

 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 52 templates are positioned ± 15 mm from the anode (red electrode). See cartoon image section “gel interpretation  9.1.6“ Each sample well is filled with 15 ml of supernatant.

Focusing
Run 2:     300 Volts – 60 mA – 12 Watts –     50  Volthours
Run 3:   1000 Volts – 60 mA – 12 Watts – 1000  Volthours

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

PGM staining
200 ml          0.2M Tris / HCl pH 8.0
 0.3 ml         4 M magnesium chloride
 0.50 g       α-D-glucose -1-phosphate disodium salt (Sigma G 7000)
 0.05  g          NADP (q.c.w. Sigma N-3886)
 0.05 g          MTT (q.c.w. Sigma M-2128)
 0.01 g          PMS (q.c.w. Sigma P-9625) (tip of the small spatula)
80  Units   Glucose-6-phosphate-dehydrogenase (q.c.w. Sigma G 7750)

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. 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. The gel can be air-dried.

Gel interpretation 
No international enzyme classification is used.
The neatest 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)