Question

Q4. Explain Bradford assay? How would you calculate protein concentration of an unknown protein sample using Bradford assay?

Answer 1

Aim: Estimation of proteins in germinating and non-geminating seeds by Bradford assay.

Principle: Bradford method is common colorimetric method The Bradford method of protein determination is based in the binding of a dye, Coomasive Blue G, to the protein. This binding shifts the absorption maximum of the dye from red to blue. The absorbance of the solution is measured at 595 nm and is proportional to protein concentration when compared to a standard curve.

Materials:

1) Bradford Reagent: Dissolve 100 mg of Coomasive Brilliant Blue G250 in 50 ml of ethanol and 100 ml of Phosphoric acid. The mixture is stirred for approximately 10 minutes. The solution is diluted to 200 ml with distilled water and filtered. Use the reagent on dilution 1:5, Reagent:DW

2) Phosphate buffer (PBS) 0.1 M, pH 7.0:

a) Dissolve 11.99 gm monobasic sodium phosphate (NaH2PO4) in 1L DW.

b) Dissolve 17.89 gm dibasic sodium phosphate (Na2HPO4) in 1L DW.

c) To prepare 0.1 M, pH 7.0 phosphate buffer, mix 39 ml of (a) and 61 ml of (b). Dilute it to 200 ml with DW.

3) Standard protein:

Bovine Serum Albumin (BSA) : Dissolve 50 mg of BSA or Gelatin powder in 20-30 ml of DW by adding 2-3 drops of 1 N NaOH. Slightly warm the solution. Make the final volume 50ml (1mg/ml).

Procedure:-

Preparation of Plant Extract:

i) Weigh exactly 1.0 gm of seed powder and geminating seeds without seed coat separately.

ii) Place the seed powder in the mortar and grind in 10 ml PBS.

iii) Homogenize the germinated seeds in 10 ml PBS.

iv) Centrifuge the content at 8000 g for 10 minutes.

v) Save the supernatant as it is the sample for protein detection. Measure the volume. Discard the residue.

    Observation table:

T.T. No.	Std. Protein (ml)	PBS (ml)	Bradford Reagent (ml)	Mix well, allow to stand for 5 min but no longer than 30 min	O.D. (595 nm)
1. Blank	0.0	2.5	5	--
2. Protein Std.	0.5	2.0	5
3. Protein Std.	1.0	1.5	5
4. Protein Std.	1.5	1.0	5
5. Protein Std.	2.0	0.5	5
6. Protein Std.	2.5	0.0	5
7. P extract (Dry Seed)	0.2	0.8	5
8. P extract (Dry Seed)	0.5	0.5	5
9. P extract (Germinating Seed)	0.2	0.8	5
10. P extract (Germinating Seed)	0.5	0.5	5

Calculations:

Plot graph of Std. Protein concentration against Absorbance. From this find out graph value for plant extract by plotting its absorbance on the straight line graph of standards.

Suppose the graph value is ‘X’ then.

                         1ml Std. protein solution = 50 µg of protein

                                                  ‘X’ ml = Y µg of protein

Plant extract taken for the analysis was 0.5 ml, hence,

            0.5 ml pf plant extract = Y µg of protein

             Total volume of plant extract = Z µg of protein

This extract prepared from 0.5 gm of plant material, hence,

                                               0.5 gm = Z µg of protein

                                              100 gm = ........? of protein

Convert this quantity of protein which in µg to gm. ( Hint: 1gm = 10,000,00 µg )

Result:

1) ……………… gm of protein/100 gm of seed powder.

2) ………………. gm of protein/100 gm of germinating seeds.

Conclusion:

Concentration of unknown = ...............mg of protein determined by assay / 0.1 ml