1. UNIVERSITI PENDIDIKAN SULTAN IDRIS
35900 TANJONG MALIM, PERAK DARUL RIDZUAN
FAKULTI :
SAINS DAN TEKNOLOGI
PRINCIPLE IN BIOCHEMISTRY (SBK3013)
EXPERIMENT 2:
PROTEIN ANALYSIS
PREPARED BY :
UMI ABIBAH BT SULAIMAN D20091034811
SITI RAHAYU BT MOHAMED NOOR D20091034855
AZMA AMIRA MOHAMAD D20091034859
NUR AFIQAH BT MUHAMAD APANDI D20091034872
AMEERA BT YAHYA D20091034814
SEMESTER 8
SESI 2009/2010
LAB SESSION
THURSDAY(10.30 A.M. - 1.30 P.M.)
LAB INSTRUCTOR
DR ROSMILA MISNAN

2. PROTEIN EXPERIMENT
Experiment 2: The determination of three protein samples using Biuret and Lowry Assay.
Objective:
1. To learn the principles of protein assays.
2. To determine protein concentrations of protein samples using the Biuret Protein Assay
and Lowry assay.
Introduction:
The determination of protein concentration is an essential technique in all aspects of
protein studies.This lab activity is designed to teach students the principles behind a common
protein estimation assay known as the BiuretProtein Assay and Lowry Protein
Assay.Although there are a wide variety of protein assays available none of the assays can be
used without first consideringtheir suitability for the application. Each method has its own
advantages and limitations and often it is necessary to obtainmore than one type of protein
assay for research applications.
In the copper ion based protein assays, protein solutions are mixed with an alkaline
solution of copper salt, cupricions (Cu2+). The protein assay is based on the interaction of
cupric ions with protein in an alkaline solution and is commonlyreferred to as the Biuret
assay. The interaction of cupric ions (Cu2+) with protein results in a purple color that can be
read at 540nm. The amount of color produced is proportional to protein concentration.
The Lowry protein assay method for protein concentration determination is one of the
most venerable and widely-used protein assays. Hydrolysis is probably the most accurate
method of determining protein concentration followed by amino acid analysis. Most other
methods are sensitive to the amino acid composition of the protein, and absolute
concentrations cannot be obtained. The Lowry procedure is sensitive, and is moderately
constant from protein to protein. The Lowry protein estimation has been so widely used that
it is a completely acceptable alternative to a rigorous absolute determination in almost all
circumstances in which protein mixtures or crude extracts are involved.

3. Result:
Biuret Assay
Table 1.1: Biuret assay
Concentration (mg/ml) Absorbance (nm)
Blank 0.00
1 0.138
2 0.139
3 0.190
4 0.192
5 0.194
6 0.211
Table 1.2: Determination using Biuret assay
Samples Absorbance Dilution Absorbance Concentration
before after dilution (mg/ml)
dilution (nm) (nm)
Chicken egg 0.936 1mL of sample + 9 mL 0.106 2.0 x 10=20
of distilled water
Quail egg 0.870 1mL of sample + 9 mL 0.090 1.6 x 10= 16
of distilled water
Duck egg 0.764 1mL of sample + 9 mL 0.066 1.0 x 10= 10
of distilled water
Chicken egg 1mL of sample + 9 mL 0.195 1.8 x 10= 18
(ayam 0.230 of distilled water
kampung)
Omega egg 0.256 1mL of sample + 9 mL 0.197 1.9 x 10= 19
of distilled water

4. Graph of absorbance value (nm) versus gelatin concentration (mg/ml) for Biuret assay
0.25
0.2
absorbance value (nm)
0.15
Y-Values
0.1
0.05
0
0 1 2 3 4 5 6 7
Gelatin concentration (mg/ml)

5. Lowry Assay
Table 2.1: Lowry assay
Concentration Absorbance (nm)
Blank 0.00
0.1mg/mL 0.053
0.2 mg/mL 0.133
0.3mg/mL 0.143
0.4 mg/mL 0.207
0.5mg/mL 0.185
0.6 mg/mL 0.166
Table 2.2: Determination using Lowry assay
Samples Dilution Absorbance Concentration
after dilution (mg/ml)
(nm)
Chicken egg 1mL of sample + 9 mL 0.072 0.16 x 10=1.6
of distilled water
Quail egg 1mL of sample + 9 mL 0.134 0.3x 10= 3.0
of distilled water
Duck egg 1mL of sample + 9 mL 0.185 0.56 x 10= 5.6
of distilled water
Chicken egg 1mL of sample + 9 mL 0.173 0.41 x 10= 4.1
(ayam of distilled water
kampung)
Omega egg 1mL of sample + 9 mL 0.131 0.25 x 10= 2.5
of distilled water

6. Graph of absorbance value (nm) versus gelatin concentration (mg/ml) for Lowry assay
0.25
0.2
Absorbance value (nm)
0.15
0.1 Y-Values
0.05
0
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7
Gelatin concerntration (mg/ml)

7. RESULT SUMMARY
Table 3.1 Protein concentration obtained from Biuret assay and Lowry assay
Type of sample Concentration (mg/ml)
Biuret Assay Lowry Assay
Chicken egg 20 1.6
Quail egg 16 3.0
Duck egg 10 5.6
Chicken egg 18 4.1
(ayam kampung)
Omega egg 19 2.5
Discussion:
In this experiment, we had used Biuret assay and Lowry assay to determine protein
concentration in five protein food samples which are chicken egg, quail egg, duck egg,
chicken egg (ayam kampung) and omega egg.
Biuret test indicate the presence of peptide bond between the amino group and the
carboxyl acid group on adjacent amino acids in a protein. The violet colour is a positive test
for the presence of protein. The greater the violet colour intense, the greater the number of
peptide bonds that reacts.
Biuret test also has its limitations and not very sensitive which are the Biuret only test
whether peptide bonds in protein are present in a sample, it will not determine how much
there is unless we compare our sample of unknown concentration with a standard of known
protein concentration.
However biuret test will not detect free amino acids and the characteristic purple
colour will not show up if we only have small peptides, since the number of peptide bonds
may not be sufficient to yield colour. The Biuret assay is not much good for protein
concentrations below about 5 mg/ml. By using the Folin‐Ciocalteu reagent to detect reduced
copper makes the Lowry assay nearly 100 times more sensitive than the Biuret reaction alone.
Based on the result obtained we had plotted the standard curve by using Biuret assays,
and we compare with the five type of albumin sample. We found that the absorbance value of

8. these five types of albumin sample is higher than the standard curve. Then the five must do
the dilution to lower the reading from the standard curve. For dilution, the ratio used is 1 ml
of protein samples to 9.0 ml of distilled water. After dilution, we get the results which are the
protein concentration in chicken egg is the highest which is 20 mg/mL, the second higher of
protein concentration by using Biuret assays is omega egg which is 19 mg/mL and the third
higher protein concentration using Biuret assays is chicken egg(ayam kampung) which is
18mg/mL. Quail egg show the protein concentration 16mg/mL and the lowest protein
concentration by using Biuret assays is duck egg which is 10 mg/mL.
Next we had carried out Lowry assay to determine the protein concentration in three
protein samples. The Lowry method is more sensitive since it combines the reactions of
copper ions with the peptide bonds under alkaline conditions 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.
The following substances are known to interfere with the Lowry assay are detergents,
carbohydrates, glycerol, potassium compounds, sulfhydryl compounds, most phenols, uric
acid, guanine, and calcium. Many of these interfering substances are commonly used in
buffers for preparing proteins or in cell extracts.
From the result of Lawry assays we also had been plotted standard curve graph, and
we compare with the five type of albumin sample. For dilution, the ratio used is 1 ml of
protein samples to 9.0 ml of distilled water. After dilution, we get the results which are the
protein concentration in duck egg is the highest among the other four which is 5.6 mg/mL, the
second higher of protein concentration by using Lowry assays is chicken egg (ayam
kampung) egg which is 4.1 mg/mL and the third highest protein concentration using Lowry
assays is quail egg which is 3.0 mg/mL. Omega egg shows the protein concentration
2.5mg/mL and the lowest protein concentration by using Lowry assays is chicken egg which
is 1.6 mg/mL.
When compare the result of Lowry assay with biuret essay, there is some differences.
This is might be because of some errors that occur while we conduct our experiment. For
instance we just estimate the volume of the sample when we poured into the vial. It should an

9. actual volume that is same for all protein. Second, there might be error when we dilute the
protein, caused by parallax error when reading the measuring cylinder.
As the conclusion, Lowry assays is best method in determining the protein
concentration because it is best used with protein concentrations of 0.01-1.0 mg/mL which is
suitable when test with 0.25 mL of protein sample of albumin of chicken, duck, quail, ayam
kampong and omega which is in the range of 0.01-1.0 mg/mL. Meanwhile, Biuret assay is not
very efficient in determining the protein concentration as it is not much good for protein
concentrations below about 5 mg/ml. Thus, albumin of duck has the highest protein
concentration, albumin of ayam kampong is the second highest in protein concentration,
albumin of quail has the third highest in protein concentration, omega chicken has the fourth
highest in protein concentration and the last one is albumin of chicken.
Instead of these two method, there are several method can be used in determining
protein concentration. First is 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. It also has
disadvantages which is the Bradford assay is linear over a short range, typically from 0 µg/ml to
2000 µg/ml, often making dilutions of a sample necessary before analysis. It is also inhibited by the
presence of detergents.

10. Conclusion
1. Protein concentration can be determined using Biuret protein assay and Lowry protein
assay.
2. The protein concentration in chicken egg is 20mg/mL , omega egg is 19 mg/mL,
chicken egg(ayam kampong) is 18mg/mL, quail egg is 16mg/mL and duck egg is
10mg/mL based on Biuret method.
3. The protein concentration in duck egg is 5.6mg/mL , chicken egg(ayam kampong) is
4.1 mg/mL, quail egg is 3.0mg/mL, omega egg is 2.5mg/mL and chicken egg is
1.6mg/mL based on Lowry method.
4. Protein concentration can be found out from the standard curve.
References
1. www.gbiosciences.com/.../633453707995878750.pdf
2. wolfson.huji.ac.il/purification/PDF/Protein.../PIERCE_BIURET.pdf
3. biochemistry.musc.edu/.../Lowry%20Protein%20Assay... - United States
4. www.molecularstation.com/protein/lowry-protein-assay/