Estimation of Proteins: 2 Methods | Plant Biotechnology

The following points highlight the top two methods used for the estimation of proteins. The methods are: 1. Lowry’s Method 2. Bradford’s (1976) Method.

1. Lowry’s Method:

This method gives a moderately constant value of proteins. Hence protein content of enzymes is usually determined by this method.

Principle:

Amino acids tryptophan and tyrosine present in proteins reduce phosphomolybdic- phospho- tungstic components in Folin- Ciocalteau rea­gent developing blue colour. This blue colour plus the colour developed by the biuret reac­tion of protein with alkaline cupric tartarate are measured by this method at 660 nm.

Requirements:

Reagents:

1. 2.0% Sodium carbonate in 0.1 (N):

NaOH—Reagent A.

2. 0.5% CuS0₄.5H₂O in 1.0% potassium:

Sodium tartarate—Reagent B.

3. Alkaline copper solution = 9 ml of A+ 1.0 ml of B:

Mixed prior to use—Reagent C.

4. Folin-ciocalteau—Reagent D:

Make a mixture of 100 g sodium tungstate (Na₂WoO₄.2H₂O) + 25.0 g so­dium molybdate (Na₂MoO₄.2H₂O) + 700 ml distilled water + 50 ml of 85% phosphoric acid + 100 ml of conc. HCl in a 1.5 litre flask and reflux gently for 10 hours. Add 150 g of Lithium sulphate + 50 ml of distilled water + a few drops of bromine water.

Boil the mixture with­out condensor to remove excess of bro­mine, cool and dilute to one litre and fil­ter. The reagent should not have any greenish tint. [Titrate with 1(N) NaOH using phenolphthalein end point to deter­mine acid concentration].

5. Stock-standard protein solution:

50 mg of Bovine serum albumin dissolved in distilled water and make the volume upto 50 ml. Add 2-3 drops of 1(N) NaOH.

6. Working standard:

10 ml of stock standard is diluted to 50 ml with distilled water (1 ml= 200 µg of protein).

7. Unknown sample-legume seeds:

Weigh 500 mg of legume seed, grind well in a mortar and pestle with 5-10 ml of buffer (used for enzyme assay). Centri­fuge and use supernatant for estimation of protein.

8. Burettes.

9. Graduated pipettes.

10. Mortar and pestle.

11. Buffer.

12. Test tubes and stand.

13. Flasks.

14. Spectronic 20.

Procedure:

1. In a series of tubes pipette out 0.1, 0.2, 0.3, 0.4, 0.5 and 1.0 ml of working stand­ard label.

2. Pipette out 0.1 and 0.2 ml of sample ex­tract label.

3. Make up the volume to 1.0 ml in all tubes with distilled water.

4. Add 5 ml of reagent C to all tubes mix well and allow to stand for 10 minutes.

5. Add 0.5 ml of reagent D, mix well, incu­bate at room temperature in dark for 30 minutes.

6. Measure the intensity of blue colour at 660 nm.

7. Plot a standard graph with concentra­tion against absorbance and find out that of unknown.

2. Bradford’s (1976) Method:

This method has a different concept. Here the capacity of a protein to bind a dye is shown.

Principle:

The binding of dye Coomassie brilliant blue G-250 to protein causes a shift in the absorp­tion maximum of the dye from 465 to 595 nm and this increase in the absorption is moni­tored at 595 nm.

Requirements:

Reagents:

1. Coomassie brilliant blue:

Dissolve 10 mg in 5 ml 95% ethanol and add 10 ml, 85% phosphoric acid and make up the volume to one litre with dis­tilled water.

2. Buffer:

0.1 (M) PO₄ buffer (pH7.0) i.e. 13.6 g K₂HPO₄ + 4.0 g KH₂PO₄. Make up the volume to one litre with dis­tilled water.

3. Sample:

Bean seed.

4. Test tubes and stands.

5. Pipettes (graduated).

6. Mortar and pestle.

7. Centrifuge.

8. Glass marking pencil.

9. Spectronic 20.

Procedure:

1. Crush 1 g bean seed with 95% ethanol centrifuge at 2000 rpm for 2-3 minutes, use supernatant as sample.

2. In a series of tubes, pipette out 0.01,0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1 ml of working standard (10-100 µg) (same as that given in Lowry’s method).

3. Pipette out 0.05 and 0.10 ml of sample in separate tubes.

4. Add phosphate buffer to make up the volume to 0.1 ml in all tubes. In tubes with 0.1 ml of working standard and sam­ple, no P0₄ buffer is added. One tube with 1 ml PO₄ as blank.

5. Add 5.0 ml of dye to all the tubes. Mix well and wait for 2-5 minutes.

The red dye turns blue when it binds to protein. Read absorbance at 595 nm between 2 minutes to one hour. Plot a graph with concentration of standard pro­tein against absorbance and find out the concentration of protein in the bean seed.