Extraction and Separation of Storage Pro­teins | Plant Biotechnology

In this article we will discuss about the requirements, reagents and procedure for extraction and separation of storage proteins.

Requirements:

1. Legume seeds/serial grains.

2. Grinder.

3. Refrigerator.

4. Centrifuge.

5. Spectronic 20.

6. Magnetic stirrer.

7. TRIS base.

8. NaCl.

9. Anhydrous Na2SO4.

10. NaOH.

11. Na-K-tartarate.

12. Na2CO3.

13. CuSO4.7H2O.

14. Folin-Ciocalteau reagent.

15. Bovine serum albumin.

16. Petroleum ether (60°-80°C).

17. Alcohol.

18. Acetone.

19. Glass column.

20. Glass wool.

21. Anhydrous sodium sulphate.

22. Balance.

23. Desiccator.

Reagents:

(i) 50 mM Tris HCl pH 8.0-1.514 g dissolved in 200 ml distilled water, adjust pH 8.0 with conc. HCl and make up the volume to 250 ml.

(ii)50 mM Tris HCl pH 8.0 + 0.5 ml NaCl dissolve in 200 ml distilled water, adjust pH 8.0 with conc. HCl and make up the volume to 250 ml.

(iii) 1 (N)NaOH-4 g NaOH in 100 ml dis­tilled water.

(iv) BSA (200 µg/ml)-20 mg BSA dissolved in 100 ml distilled water.

(v) Alkaline copper tartarate:

(a) First dissolve 10 g sodium carbonate and 2 g NaOH in 100 ml water [10% Na₂CO₃ in 0.5(N) NaOH],

(b) 2% CuS0₄.5H₂0-Dissolve 1 g CuS0₄.5H₂0 in 50 ml distilled water.

(c) 2% Na-K tartarate-Dissolve 1 g in 50 ml distilled water.

(d) 0.5 ml of (b) and (c) each, add to 10 ml of (a).

(vi) Folin Ciocalteau reagent (1:10) 5 ml in 50 ml distilled water.

Procedure:

Defatting seed meal:

1. Grind legume seed to a fine powder, pack 20 g powder in a glass column over anhydrous sodium sulphate and place glass wool over it.

2. Wash this seed meal column four times with petroleum-ether and the fifth time with acetone.

3. After drying invert the column, weigh the defatted seed powder and keep in a des­iccator.

Extraction of protein:

4. Take one gram of the defatted seed pow­der in a beaker and add 50 mM Tris HCl (pH 8.0). Keep the beaker on a mag­netic stirrer for this at 4°C.

5. Centrifuge for 15 minutes at 3000 rpm and measure the volume of the supernatant. This is the albumin fraction- A.

6. Add 10 ml of 50 mM Tris HCl (pH 8.0) + 0.5(M) NaCl to the residue, stir on a magnetic stirrer for one hour at 4°C.

7. Centrifuge for 15 minutes at 3000 rpm and measure the volume of the super­natant. This is globulin fraction-B.

8. Add 10 ml of 70% alcohol to the resi­due, stir on a magnetic stirrer for one hour at 4°C.

9. Centrifuge for 15 minutes at 3000 rpm and measure the volume of supernatant. This is prolamine fraction-C.

10. Repeat the process with the residue by adding 10 ml of 1 (N) NaOH. This is glutelin fraction-D.