PRX

Melon

PRX – (Peroxidase)

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 3-6 (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
0.5 g ammonium persulfate (q.c.w. Sigma A- 3678) in 5.0 ml distilled water.
Store in refrigerator for one week

0.5 v/v % Sepalyte (pH 3-6 (Cat. No.42006)
2.5 ml Sepalyte (pH 3-6 (Cat. No.42006)) 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

Citrate HCl  0.063 M pH 4.4
Bring 37.0g  trisodium citrate dihydrate  (q.c.w. Sigma C-7254) in ± 1800 ml distilled water. Stir until fully dissolved. Adjust the pH to 4.4 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.

O-Dianisidine
2.7 g O-dianisidine (Sigma D-3252) in 500 ml methanol (q.c.w. BDH 29192). Keep the solution in the dark. Store in refrigerator.

50 %  v/v %  H2O2   (see instructional note)
Available at general drugstore.  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 acrylamide 29: 1 solution
1.0 ml        Sepalyte pH 3-6
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 light. Store the gels in a sealed bag in the refrigerator for up to 2 weeks.

Sample preparation
Seeds are germinated for 3 or 4 days in towels or  “harmonica”-shaped filter paper at ± 30 ° C and in the dark.   Separate the hypocotyledon/roots from the kernel and bring the hypocotyledon/roots into a 96-well microplate (try to keep sample size consistent). Flatten the hypocotyledons/roots onto the bottom of the well. FREEZE for at least 2 hours or better overnight. After the freezing step, the microplate + frozen samples have to be homogenized, using the Terminator, immediately after taking them from the freezer.  Crush for three minutes after adding 150 ml of 0.5% Sepalyte pH 3-6 (Cat. No.42006) extraction fluid to each well. Centrifuge for 10 minutes at 3000 rpm. at 10° C  (or at room temperature). Repeat centrifugation if time permits.

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
Run 1:   600 Volts – 60 mA – 10 Watts – 100 Volthours

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:     200 Volts – 60 mA – 10 Watts –    100  Volthours
Run 3:   1000 Volts – 60 mA – 10 Watts – 1000  Volthours

PRX staining
200  ml    0.063 M citrate – HCl pH 4.4
25  ml     O-dianisidine solution
0.60  ml     50 %  H2O2 

Add the reagents together and mix until fully dissolved and stain the gel at room temperature until the orange 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 be air-dried.     

Gel interpretation
Interpret the bands of interest.

The first band of interest from the anode is genotyped  “11”
The furthest 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.

This is an illustrative cartoon image and does not picture the real PRX profile.

ProTec Bioseparation (Protein Electrophoresis)