2012

Protein Estimation Methods

Sundaram
Bharathidasan University,Trichy
22/1/2012

ESTIMATION OF PROTEIN BY LOWRY METHOD

Aim:
To estimate the amount of serum protein present in the given sample.

Principle:
The carbonyl group of protein molecules reacts with copper and potassium of Lowrys reagent to give a blue colored compound. This complex together with tyrosine and phenolic compound present in the protein sample reduce the phospho molybdate of Folins phenol reagent.

Reagents:
1. Lowrys Reagent:
Solution A: 2% Na2CO3 in 0.1 N NaOH. Solution B: 0.5% CuSO4 in Rochelles Salt. (Sodium Potassium Tartarate) Mix the solutions A & B in 50:1 ratio before use.

2. Stock Standard of protein:

10 mg of BSA is dissolved in 10 ml of distilled water.

3. Working Standard Solution:

1ml of the stock solution is distilled to 10ml in distilled water.

Procedure:
Prepare various concentrations of Folins phenol ranging from 20-100gby pipetting out 0.2, 0.4, 0.6, 0.8 & 1.0 ml of Folins phenol in a series of test tubes and make up to 1ml with distilled water. Label the test tubes S1 to S5 respectively. Maintain a blank with 1ml of distilled water and label it as B. Dilute 0.1 ml of serum to 20 ml. From this, take 0.1ml for test in two test tubes marked as T1 & T2 respectively. To all the test tubes add 4.5 ml of Lowrys reagent and allow it to stand for 10 minutes. To all the test tubes add 0.5 ml of Folins phenol and incubate in dark for 20 minutes. Read the color developed at 620 nm against reagent blank. Draw a standard graph by plotting concentration of plotting concentration of protein in X axis & absorbance in Y axis.

Concentration calculation:
10 ml of the stock solution contains 10 mg of BSA. 1 ml of the stock solution is diluted to 10 ml of working std. solution. 1 ml of the stock solution contains 1 mg of BSA. Therefore, 10ml of working std. contains 1 mg of BSA. 0.2 ml of working standard contains (0.21) /10=0.02 mg =20 g/100 ml

Incubate at room temperature for 10 min

Particulars

B S1 S2 S3 S4 S5 T1 T2

Vol. of Conc. of Vol. of test Vol. of Vol. of working working sample(ml) dis.water Lowrys std. std. reagent(ml) soln.(ml) soln.(g) 1 0.2 20 0.8 0.4 40 0.6 0.6 60 0.4 4.5 ml 0.8 80 0.2 1.0 100 0.1 0.9 0.1 0.9

0.5 ml

Unknown calculation:
_____O.D corresponds to _____g of protein. 0.1ml of diluted serum contains_____ g of protein. 10ml of dil. serum contains 10( )/0.1=_____ g of protein. 10ml of dil. Serum is derived from 0.5 ml of serum by 1 in 20 dilutions. 0.5ml of serum contains_____ g of protein.

Result:
The amount of protein present in 100 ml of the given serum sample is found to be_______g

Incubate in dark for 20 min

Vol. of Folins phenol

O.D at 620 nm

ESTIMATION OF PROTEIN BY BRADFORDS METHOD

Aim:
To estimate the amount of protein in the given sample by Bradfords method.

Principle:
The Bradfords assay, a colorimetric assay, is based on an absorbance shift in the dye Coomasie by the binding of protein. During the formation of this complex, two types of bond interaction takes place. The red form of Coomasie dye first donates its free proton to the ionizable group on the proteins which causes disruption of the proteins native state, consequently exposing its hydrophobic packets. These packets on the proteins tertiary structure bind non-covalently to the non-polar region of the dye via vanDerwaals forces, positioning the positive amino groups in proximity with the negative charge of the dye. The bond is further strengthened by the ionic interaction between the two. Binding of the protein stabilizes the blue form of Coomasie dye, thus the amount of complex present in the solution is a measure for the protein concentration by use of an absorbance reading. The amount of the dye binding to different molecules varies and hence requires a careful selection of protein standard.

Reagents:
1. Bradford s reagent:
100 mg of CBB G250 is dissolved in 50 ml of 95% ethanol and concentrated orthophosphoric acid is added and make up to 200 ml with distilled water and stored at 4C.

2. Stock standard of BSA:

10 mg of BSA is dissolved in 10 ml of distilled water.

3. Working standard solution:

1ml of the stock solution is diluted to 10 ml with distilled water.

Procedure:
Prepare various concentrations of protein standards ranging from 10-100 g by pipetting 0.2, 0.4, 0.6, 0.8 & 1.0 ml of working standard solution into a series of test tubes and make up to 1ml with dis. Water. Label the test tubes S1 to S5 respectively. Maintain a blank with 1ml of distilled water and label it as B. Similarly take 0.1 ml of serum in two test tubes marked as T1 and T2 respectively. Add 5 ml of the dye solution in each test tube and make up to 6ml. Mix the contents well and allow standing for 5 minutes. Read the absorbance at 595 nm against reagent blank. Draw a graph by plotting concentration of protein in X axis and absorbance in Y axis

Concentration calculation:
10 ml of the stock solution contains 10 mg of BSA. 1 ml of the stock solution is diluted to 10 ml of working standard.

1 ml of the stock solution contains 1 mg of BSA. Therfore, 10 ml of working std. contains 0.21/10=0.02 m g =20g

Particulars

B S1 S2 S3 S4 S5 T1 T2

Vol. of working std. soln(ml) 0.2 0.4 0.6 0.8 1.0 -

Conc. of working std. soln(ml) 20 40 60 80 100 -

Volume of Volume of test dis.water(ml) sample(ml) 0.1 0.1 1 0.8 0.2 0.4 0.2 0.0 0.9 0.9

Vol. of Incubation Bradfords period reagent(ml) Mix the contents and allow to stand for 5 min

O.D at 595 nm

5 ml

Calculation:
________O.D corresponds to ______g of protein. 0.1 ml of serum contains ______ g of protein. 100 ml of serum contains 100( )/0.1=______ g of protein.

Result:
The amount of protein present in 100 ml of given serum sample is found to be______g Normal value:6-8 g/100 ml of blood.