Construction of Protein Standard Curve
Lowry’s assay for total protein is one of the most commonly performed colorimetric assays. This procedure is sensitive because it employs
two colour forming reactions. It uses the Biuret reactions in which Cu2+
in presence of a base reacts with a peptide bond under alkaline conditions resulting in
reduction of cupric ions (Cu2+) to cuprous ions (Cu+), and lowry’s reaction in which the Folin’s
Ciocaltaeu reagent which is a mixture of sodium tungstate, sodium
molybdate and phosphate, along with copper sulphate solution when mixed with the protein,
a blue purple colour is produced which can be assesed by measuring
the absorbance at 650-700nm. The blue purple colour is formed due to the
reduction of phosphomolybdotungstate to hetero-polymolybdenum blue by the copper catalysed oxidation of
aromatic amino acids tryptophan and tyrosine . Thus the intensity of color depends on the
amount of these aromatic amino acids present and will thus vary for different proteins. Most protein estimation techniques use Bovine
Serum Albumin (BSA) as a standard protein, because of its easy availability and low cost
with improved purity. Materials Required
Standard Protein solution [200ug/ml]. Alkaline copper reagent.
Folin’s Ciocaltaeu reagent. Colorimeter
Pipettes. Procedure Arrange the reagent solutions prepared, on
the table. Label the test tubes with the volume taken
and arrange them in a test tube rack . Pipette out the standard protein solution
from the standard flask into the test tubes labelled [0.2ml-1ml]
Pipette a known volume of the unknown solution to the tube labelled “unknown” arranged
in the test tube rack . To the test tube labelled ‘Blank’ , add
1ml of distilled water using a micropipette. Volume in each test tube is made up to 1 ml
by adding distilled water. Add 5ml of alkaline copper reagent to all
the test tubes. Vortex and Incubate for 10 minutes at room temperature.
The solution in all the test tubes has turned blue in colour.
After incubation, add 600ul of Folin’s Ciocalteau reagent to all test tubes using micropipette.
Vortex and Incubate for 20 minutes at room temperature.
After incubation, the color intensity varies accordingly with the concentration of protein
present in the tubes . Now record the absorbance of each solution
at 650 nm using a colorimeter. Plot the absorbance against amount of protein
in milligrams to get a standard calibration curve. Check the absorbance of unknown sample
and determine the concentration of the unknown sample from the standard curve plotted.