Construction of Protein Standard Curve(Amrita University)

Uploaded by amritavlab on 22.09.2011

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.
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.