Urea Soluble Protein

Tomato

UPS – Urea Soluble Protein (isoelectric focusing)

8 M Urea
Bring 24 grams of urea (Sigma U-1250) and 30 ml distilled water in a beaker. Mix well and bring to a final volume of 50 ml with distilled water. Do not store.

Sepalyte pH 5-6 (Cat. No. 42014)
Ready to hand Sepalyte Solution (ProTec Bioseparation). Store in refrigerator.

Acrylamide /bis acrylamide 58:  1 solution
Sepalyte 5-6, Mix 5.0-ml acrylamide/bis acrylamide 29: 1 sol.  (Sigma A-3574) with 5.0 ml distilled water.  Add 1.45-g acrylamide (Sigma A-3553). Stir until fully dissolved. Store in refrigerator.

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

0,1% w/v riboflavin
0.05 g riboflavin (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 (Sigma A- 3678) in 5.0 ml distilled water.
Store in refrigerator for one week

Diluted hydrochloric acid ( 1: 20)
0.5 ml concentrated HCl (BDH 28507) + 9.5 ml water.

USP extraction
Bring 36 g urea (Sigma U-1250), 7.5 g sucrose (Sigma S-8501), 0.08 g glycine (Sigma G-7126), and 0.120 g Tris (Sigma T-8524) in a beaker. Add  ± 60 ml distilled water. Stir until fully dissolved. Adjust the pH to 7.9 with the diluted hydrochloric acid. Add 0.1 g dithiotreitol (Sigma D-9163) to the solution. Bring to a final volume of 100 ml with distilled water. Add a trace (tip of a small spatula) of Orange G  (Sigma O-1625). Do not store.

Cathode solution- 0.6 M glycine
11.25 g glycine (Sigma G-7126) in 250 ml distilled water. Add a trace (tip of a small spatula) of bromphenol blue (Sigma B-8026). Store in refrigerator.

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

Trichloroacetic acid( TCA) 20 %  w/v
Bring 400 g of trichloroacetic acid (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 (Sigma B-7920) in 200 ml ethanol 96%. Stir until fully dissolved. Add 250-ml water and 50 ml 100 % acetic acid (BDH27013) to the solution. Mix well before use. Store at room temperature.

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

Destain solution
Bring together 200 ml acetic acid 100% (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 working solution and 250 ml destain solution. Do not store.

Gel preparation
9.5 ml        8M urea solution
2.0 ml        Acrylamide/bis 58:1 solution
1.0 ml        Sepalytes 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 at room temperature and use the gel within 24 hours.

Sample preparation
Turn the cooling supply on and set at a temperature of ± 17° 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 two 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 lines in the gel. Blotting paper wicks (1 x 6-x 260 mm) are used. Wet the cathode wicks with cathode fluid 6 and the anode wick with anode fluid 3. Gently blot the wicks but keep them fairly wet. Place the cathode wicks onto the cathode prints in the gel and the anode wick onto the anode print (in the middle). On top of the wicks, place the electrode plateau.

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

Prefocusing
Run 1: 600 V–30 mA–15 W–300 V/h

Sample application
After the prefocusing step, the clear 52 or black 96 well templates are positioned ± 20 mm from the anode (red electrode). See cartoon image section “gel interpretation “ Each sample well is filled with 15 µl of supernatant for the 52 well templates and 8 µl of supernatant when using the 96 well templates.

Focusing
Run 2: 500 V–30 mA–15 W–100 V/h
Run 3: 2000 V–30 mA–15 W–1500 V/h

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, swirls 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.

 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)