To perform protein estimation by Lowry’s method.

AIM: to perform protein estimation by Lowry’s method.

PRINCIPLE:
Lowry’s method of protein estimation is the most widely used and accepted method for accurate determination of protein concentration. The method is a combination of biuret reaction and folin-ciocalteau reaction. In the first step of the reaction protein binds to copper in alkaline medium and produces Cu++ In the second step, Cu++ catalyses oxidation of aromatic amino acid by reducing phosphomolybdotungstate to heteropolymolybdanum blue. This reaction produces strong blue color, which predominantly depends upon tyrosine and tryptophan content of protein and to a lesser extent cysteine and other residues in protein.

PROCEDURE:
1) Pipette standard BSA (0.5 mg/ml) and test sample as given in table in duplicates.
2) Adjust the volume to 0.2 ml with distilled water.
3) Add 3 ml of complex forming reagent, mix and keep for 10 minutes.
4) Add 0.3 ml of solution III. Mix thoroughly and incubate for 20-30 minutes at room temperature.
5) Read the optical density on spectrophotometer at 660 nm or on a colorimeter using suitable filter and record the readings.
6) Plot a calibration curve by taking optical density reading on ‘y’ axis against std. Protein concentration (in mg) on ‘x’ axis.
7) Record the value X from the graph corresponding to the optical density reading for the test sample.
8) Calculate the protein concentration using the following formula.

Protein concen. In test sample = X/V (mg/ml)
Where X = value from the graph in mg.
V = volume of the sample in ml.

NOTE:
To prepare complex forming solution mix 0.5 ml of solution I and 50 ml of solution II just before use.
OBSERVATION TABLE:

RESULT:

Protein estimation by Barford’s method.

Protein estimation by Barford’s method.

PRINCIPLE:
It is a rapid and accurate method for estimation of protein concentration. When compared to Lowry’s method, it is subjected to less interference by common reagents and non-protein components of biological sample.
The assay relies on the binding of the dye Coomassie blue G250 to protein. The quantity of protein can be estimated by determining the amount of dye in the blue ionic form. This is usually achieved by measuring the absorbance of the solution at 595nm or 625nm.

PROCEDURE:
1) Pipette standard BSA (0.5mg/ml) & test sample as indicated in table in duplicates.
2) Adjust the volume to 0.2ml with distill water.
3) Add 3ml of Barford’s reagent & mix it thoroughly. Incubate at R.T. for 10 min.
4) Read the optical density on spectrophotometer at 595nm/625nm or on a Colorimeter using a suitable filter & record the reading.
5) Read the optical density on spectrophotometer at 595 nm/625nm or on a Colorimeter using a suitable filter and record the readings.
6) Construct a calibration or standard curve by plotting avg. optical density reading on ‘y’ axis against std. Protein concentration on ‘x’ axis.
7) Calculate the concentration in the test sample using the following formula.

Protein conc. In test sample = X/V mg/ml
Where, X= value from graph in mg V= volume of sample in ml

Result: - From the Std. Curve, determine & Report Conc. Of protein in the test sample.

To perform the estimation of protein by Biuret method

AIM: To perform the estimation of protein by Biuret method.

PRINCIPLE:
Biuret method is the simplest method for protein estimation. This method is sensitive to the amino acid composition of the protein. Its sensitivity is moderately constant from protein to protein and because of its simple procedure and quick result, it is used to estimate protein in crude extract over a large range of concentration. This method can also be used to monitor the concentration of protein during purification.
This assay is based on copper ions binding to peptide bonds of protein under alkaline conditions to give a violet or purple color. The intensity of the charge transfer absorption bond resulting from the Cu-protein complex is linearly proportional to the mass of protein present in the solution. The chromophore or light-absorbing center seems to be a complex between the peptide backbone and cupric ions.

PROCEDURE:
1) Pipette standard BSA (50 mg/ml) and test sample as indicated in table in duplicates.
2) Adjust the volume to 0.2 mi with distilled water.
3) Add 3 ml of Biuret reagent. Mix and incubate at Room Temperature for 10 minutes.Read the optical density using spectrophotometer at 600 nm or colorimeter using suitable filter and record the readings.

1) Construct a calibration curve by plotting average optical density reading on ‘Y’ axis against std. Protein concentration (in mg) on ‘X’ axis.
2) Record the value ‘X’ from the graph corresponding to the optical density reading for the test sample.
3) Calculate the sample concentration using the following formula.
Protein concentration in test Sample=(X/V)x1000 mg/ml

X= Value from graph in mg
V= Volume of Sample in ml
RESULT: