Biuret Bradfod Lowry

Convenience -very high, -more convenience -fairly convenience

independent of than lowry method [involve two steps and
composition amino [simpler, faster, time consuming]
acid inexpensive and more -can be performed at
-involves single sensitive thus more room temperature
incubation of 30 accurate] -double incubation of
minutes -compatible with wide 40 minutes. The first
-good general protein range of substances incubation is 10
assay for batches of -dependent on the minutes, second
material for which amino acid incubation is 30
yield is not a problem. composition of the minutes.
measured protein.
-single incubation of 5
minutes
Sensitivity -least sensitive than -more sensitive than -sensitive to low
bradford method and lowry method protein concentration
lowry method. -1-100 microgram/mL -sensitive over a wide
-1-10 mg/mL of protein of protein range
concentration - 20-300microgram/mL
-dependent on of protein
composition of amino
acid
Generality -its’ consistency is -it’s consistency is Its’ consistency is
quite fair good excellent
Linearity -Linear -non-linear -non-linear
concentration concentration
dependence dependence

Discussion

Protein determination can be carried out by using spectrophotometer. Spectrophotometer can

measure the amount of light that a sample absorbs. The analyte absorbs photons contained in the
light beam thus reducing the intensity of photon in the light beam. The higher the protein
concentration, the intensity of photon in the light beam reduces more.

To determine the protein, I apply biuret protein assay, Bradford protein assay and lowry protein
assay. In biuret protein assay, there are two samples prepared. First is the reference (blank) that
contains biuret reagent. Each time the sample (biuret reagent + BSA (bovine serum albumine) put in
the spectrophotometer to obtain optical density (absorbance), the blank will be put first as it is act
as a reference. A proper reference solution contains colour reagent plus sample buffer. The
difference between the reference and a sample is that the concentration of the assayable substance
in the reference solution is zero.

Each time BSA is added into the reagent, it must be incubated. The incubation process is to
make sure the solution mix well. Biuret method is a test employed to detect the presence of
peptide bonds. The presence of peptide bonds can be depicted by the changes of colour
from blue to violet-colored complex. To assay the protein concentrations, the biuret method
is used as peptide bonds happen with the same frequency per amino acid in the peptide.
According to the Beer-Lambert law, the intensity of the color, and hence the absorption at
540 nm, is directly proportional to the protein concentration.

Bradford method purpose is also the same as biuret method. However, this method is much
more popular as it is simpler, faster, inexpensive and more sensitive thus more accurate
than the the biuret and lowry method. The Bradford assay, a colorimetric protein assay, is
based on an absorbance shift of the dye Coomassie Brilliant Blue G-250 in which under
acidic conditions the red form of the dye is converted into its bluer form to bind to the
protein being assayed. The binding of the protein stabilizes the blue form of the Coomassie
dye; thus the amount of the complex present in solution is a measure for the protein
concentration, and can be estimated by use of an absorbance reading. Lowry method is not
as good as Bradford method however the sensitivity of lowry method towards protein
concentration is very good compare to biuret method. The method combines the reactions
of copper ions with the peptide bonds under alkaline conditions (the Biuret test) with the
oxidation of aromatic protein residues. The Lowry method is best used with protein
concentrations of 0.01-1.0 mg/mL. and is based on the reaction of Cu +, produced by the
oxidation of peptide bonds, with Folin-Ciocalteu reagent (a mixture of phosphotungstic acid
and phosphomolybdic acid in the Folin-Ciocalteu reaction). The reaction mechanism is not
well understood, but involves reduction of the Folin reagent and oxidation of aromatic
residues (mainly tryptophan, also tyrosine). The concentration of the reduced Folin reagent
is measured by absorbance at 750 nm[3]. As a result, the total concentration of protein in the
sample can be deduced from the concentration of Trp and Tyr residues that reduce the Folin
reagent. The method was first proposed by Lowry in 1951

Conclusion :

From the experiment, I can conclude that, Bradford method is the most convenient method
amongst biuret and lowry. This is because it act fast, simple, very sensitive thus give accurate
reading.