1. INTRODUCTION
The quantitation of protein content is important and has many applications in clinical
laboratory practices and in research especially in the field of biochemistry. The accurate
quantitation of protein content is a critical step in protein analysis. Over the past two decades,
different protein assay techniques have been developed for the assessment of the protein
concentration in a sample. Spectroscopic methods are the most common way to quantitate
protein concentrations. UV-Vis Spectroscopy is primarily used for quantitative analysis in
chemistry and one of its many applications is in protein assays. Classical methods such as the
biuret test, Bradford test, spectrophotometric assay at 280 nm, Smith test, and Lowry test are
some of the most commonly used techniques in protein quantitation.
Given the importance of protein assay, it is significant to choose the appropriate
technique from the available methods. In doing this, several factors such as the nature of the
protein, the nature of other components present in the sample, and the preferred speed,
accuracy, and sensitivity of assay are considered. It is also of great importance to know which
particular range of protein concentration an assay is sensitive to. In an ideal assay, the most
preferred calibration curve generates a linear response to the standard solutions that covers the
range of the concentration of the unknown. As the linearity range for the calibration curve is
known, it will make the assay more accurate, time efficient and cost effective. Biuret test is of
particular interest in this study.
Quantization of the total protein content in a sample is a critical step in protein
analysis. Molecular UV absorption spectroscopy is very efficient in quantitative analysis such
as protein quantization and has extensive application in chemical and clinical laboratories
worldwide. It is also of great importance to know which particular range of protein
concentration an assay is sensitive to. In an ideal assay, the most preferred calibration curve
generates a linear response to the standard solution that covers the range of the concentration
of the unknown. As the linearity range for the calibration curve is known, it will make the
assay more accurate.
However, similar with other protein assays, the linear range for the Biuret test found
in different literature varies. The most common lover limit of the calibration curve for the
Biuret test is 1mg/sample. The study about the lowest concentration of the linear range for
Biuret test is aim to:
 Determine the sensitivity of the protein quantitation technique
 Verify the range of protein concentration at which the method for protein
quantification is accurate
 Provide the protein concentration range in which it will generate the best standard
calibration curve
Besides, in performing total protein assays, there are five issues of concern:
(1) Sensitivity and techniques of the method
(2) Clear definition of units
(3) Interfering compounds
(4) Removal of interfering substances before assaying samples
(5) Correlation of information from various techniques

2. TITLE
Estimation Of Protein (Biuret Assay Method) From Supplied Sample
APPARATUS
Beakers, Test tube holder, Micropipette, Beakers, Spectrophotometer, Water bath, 50ml
volumetric flask
MATERIALS
Bovine serum albumin solution (BSA), Phosphate buffer, Bradford reagent, Unknown protein
supplied sample
PROCEDURES
(1) 0.0, 10.0, 20.0, 40.0, 60.0, 80.0 and 100.0 µl of Bovine Serum Albumin (BSA)
solutions are measured using micropipette and filled in different test tubes. The 7
solutions are made up to 100µl by phosphate buffer solution.
(2) 5ml of Bradford reagent is added into each test tube and mixed well. The solutions
are placed in spectrophotometer at 595nm for test. The reading of the solutions are
taken and recorded. A graph of the absorbance at 585nm against the reagent blank is
plotted.
(3) An unknown supplied sample is pipette into 50ml volumetric flask. Distilled water is
added to the volumetric flask and make up to 50ml. 100µl and 200µl of the solution is
pipette into 2 different test tubes and repeated with the above procedure.
Spectrometer Micropipette

3. RESULTS AND CALCULATIONS
Table of different content of BSA solutions in different test tubes
No. of test
tubes
Volumes of
BSA (µl)
Weight of
BSA (µg)
Volume of
phosphate
buffer (µl)
Optical
Density (A)
(595nm)
Volume of
Bradford
reagent (ml)
1 0.00 0.00 100.00 0.000 5
2 10.00 1.00 90.00 0.107 5
3 20.00 2.00 80.00 0.140 5
4 40.00 4.00 60.00 0.272 5
5 60.00 6.00 40.00 0.418 5
6 80.00 8.00 20.00 0.555 5
7 100.00 10.00 0.00 0.695 5
Table of unknown protein concentration food sample
No of test tubes Volumes of food
sample (µl)
Optical Density (A)
(595nm)
Volume of Bradford
reagent(ml)
1 100 0.305 5
2 200 0.615 5
Concentration of BSA solution prepared = 100𝜇𝑔/𝑚𝑙
=
100µ𝑔
1000𝜇𝑙
= 1𝜇𝑔/10𝜇𝑙(as shown in the x-axis of the
graph)
Concentration of 100µl of unknown food sample
1ml= 1000µl
100µl = 4.40µg
(100µl × 10 ) = (4.40 ×10 )µg
1000µl = 44.00µg/ml
*There is 44.00µg/ml in term of concentration of protein found in 100µl unknown food
sample.
Concentration of 200µl of unknown food sample
1ml= 1000µl
200µl = 8.85µg
(200µl × 5 ) = (8.85 ×5 )µg
1000µl = 44.25µg/ml
*There is 44.25µg/ml in term of concentration of protein found in 100µl unknown food
sample.

4. DISCUSSION
In this experiment, the concentration of unknown food supplied sample found is
almost similar, that is 44.00µg for 100µl food sample and 44.25µg for 200µl food sample.
Both food samples come from the same solution hence their concentration should be almost
the same. The differences between these 2 solutions maybe causes by the percentage error of
spectrophotometer or the human error when preparing the solutions. Besides, all readings
should be taken within 10 minutes as with most assays, the Biuret can be scaled down for
smaller cuvette sizes, consuming less protein. Proteins with an abnormally high or low
percentage of amino acids with aromatic side groups will give high or low readings,
respectively.
Protein contains tyrosine and tryptophan side chains that are fairly strong absorbers of
light at the ultraviolet region. Consequently, after suitable dilution to produce on scale
absorbance readings, total proteins can be estimated from UV absorbance spectra.
Bradford assay is based on the binding specificity of the dye Coomassie Brilliant
Blue-G250 for protein molecule but not for other cellular constituents. This organic dye binds
specifically to the tyrosine side chains. The binding of the dye to protein shifts the peak
absorbance of the dye. Unbound Coomassie Blue absorbs light maximally at wavelength of
465nm, while the absorption maximum is at 595nm when the dye is bound to protein. The
absorbance of light by the dye protein complex at 595nm is proportional to the amount of
protein bound (over a limited range); i.e. there is a linear relationship between absorbance and
the total protein concentration of the sample over a narrow range. In the copper ion based
protein assays, protein solution are mixed with an alkaline copper salt, cupric ions (Cu2+
).
Under alkaline conditions, cupric ions (Cu2+
) chelate with the peptide bonds resulting in
reduction of cupric ions (Cu+
).
Spectrophotometer is employed to measure the amount of light that a sample absorbs.
The instrument operates by passing a beam of light through a sample and measuring the
intensity of light reaching a detector. The beam of light consists of a stream of photons. When
a photon encounters an analyte molecule, there is a chance the analyte will absorb the photon.
This absorption reduces the number of photons in the beam of light, thereby reducing the
intensity of the light beam. Hence, this is why spectrophotometer is used to detect the
concentration of protein in an unknown sample.
CONCLUSION
In this experiment, I had found that the concentration of protein in 100µl of unknown food
sample is 44.00µg/ml while for 200µl is 44.25𝜇g/ml.
REFERENCES
(1) Boyer, R. (2000) Modern Experimental Biochemistry, 3rd
edition; Addison
Wesley Longman
(2) Gornall, G. Bardwill (1949) Determination of Serum Proteins by means of the Biuret
Reaction
(3) Harris (2003) Quantitative Chemical Analyse, 6th
edition; W.H. Freeman and Company