PRS

Squash

PRS – (Protein from Seed)

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 5-6 (Cat.No. 42014 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  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

Trichloroacetic acid( TCA) 20 %  w/v
Bring 400 g of trichloroacetic acid (q.c.w. Sigma T-4885) and 1500 ml water in a beaker. Stir until fully dissolved. Bring to a final volume of 2000 ml with distilled water.
Store at room temperature.

Coomassie stock solution
Bring 1.0 g brilliant blue (q.c.w. Sigma B-7920) in 200 ml ethanol 96%. Stir until fully dissolved.  Add 250-ml water and 50 ml 100 % acetic acid (q.c.w. BDH 27013) to the solution. Mix well before use. Store at room temperature.

Destain solution
Bring together 200 ml acetic acid 100% (q.c.w. BDH 27013), 800 ml ethanol 96% (or methanol 100%) and 1000 ml distilled water. Store at room temperature

Coomassie working solution
Bring together 50 ml fresh stock solution and 250 ml destain solution. Do not store.

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

Gel preparation
9.5 ml        16 % glycerol
2.0 ml        Acrylamide/bis acrylamide 29: 1 solution
0.3 ml        Sepalyte pH 3-10
0.7 ml        Sepalyte pH 5-6
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 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 microplate. Crush the endosperms using the ISOLAB crusher and rubber hammer. To each well 200 ml 0.5 % Sepalyte  (pH 3-6 (Cat. No.42006) 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 75μl of supernatant to a new, clean 96 well microplate and centrifuge 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 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 – 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 µl of supernatant.

Focusing
Run 2:     200 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.

Coomassie staining

  • Pour on the gel ±300 ml of 20% TCA solution. Let the proteins precipitate for 5 minutes. After the 5 minutes of fixation, swirl the tray for 15 minutes.
  • Rinse off the TCA, using dH2O.
  • Add   ± 300 ml Coomassie working solution to the gel. Stain until blue bands can be clearly visualized.
  • Rinse off the blue stain using dH2O, destain (if necessary) with the destaining solution. 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.


ProTec Bioseparation (Protein Electrophoresis)