Table of Contents

Table of Contents..................................................................................................... 1
Abstract....................................................................................................................2
1.0 Introduction .................................................................................................................. 3
2.0 Objectives ..................................................................................................................... 3
3.0 Theory............................................................................................................................4
4.0 Procedures..................................................................................................................... 5
5.0 Apparatus... 6
6.0 Results............................................................................................... 6
7.0 Sample Calculations...................................................................................................... 9
8.0 Discussions 11
9.0 Conclusions... 13
10.0 Recommendations....................................................................................................... 13
11.0 References................................................................................................................... 13

Abstract
Protein consist long chains of amino acid that are folded together in their structure to form
more complex of molecules. The amino acid will through the process of protein folding with
are primary, secondary, tertiary and quaternary structure. Characteristic of proteins allows
their ability to bind other molecules specifically and tightly. The objective of this experiment
is to determine protein concentrations using three different assays with are lowry reagent,
Bradford reagent and standard curve 280nm. Bovine serum albumin (BSA) and gelatine will
be used to act as protein sample in this experiment. The Lowry method is routinely used for
determining the protein content of biological samples. In this method, protein is first treated
with alkaline copper sulfate in the presence of tartrate, followed by the addition of folin
phenol reagent. Then, bradford assay is highly time-sensitive, with precipitation of protein
occurring about 10 min after contact. It is fairly accurate and samples that are out of range can
be retested within minutes. The Bradford assay has become the colorimetric method of
choice, owing principally to its high sensitivity, perceived linearity, and the speed of analysis.
The experiment is started with preparation of both protein sample with are bovine serum
albumin (BSA) and gelatine. The, it will continuously with preparation of reagent; lowry,
Bradford reagent and standard curve. Lastly, both protein samples will analysis with that three
of the reagents.
1.0 Introduction
Protein consist long chains of amino acid that are folded together in their structure to form
more complex of molecules. The amino acid will through the process of protein folding with
are primary, secondary, tertiary and quaternary structure. Many proteins can fold unassisted,
with due to the chemical properties of their amino acids but certain unfolded protein required
the molecular reaction to fold. Proteins will fold into 3-dimensional structures at the
quaternary structure when the protein folding process is completely.
The shape into which a protein naturally folds is known as its native conformation.
Protein with bricks and mortar of cells, playing structural and functional roles with are
interacting each other as well as with other biomolecules such as DNA or RNA (Fornes et al.,
2014). Characteristic of proteins also allows their ability to bind other molecules specifically
and tightly. The individual amino acid residues are bonded together by peptide bonds and
adjacent amino acid residues.
When protein forms primary structure, amino acid will connect in linear chain called
polypeptides. Then, when hydrogen bonding between molecules are react to each other, the
long chains of amino acid will form alpha helix sheet at secondary structure (Fornes et al.,
2014). During tertiary structure, protein that in alpha helix sheet will fold together and start to

become complex. These tertiary structures will controls the basic function of the protein.
While, quaternary structure is non-covalent that build up when folded protein combine
together. Example of quaternary structure is haemoglobin that has two alpha globin and two
beta globin polyproteins
2.0 Objectives
(i) To determine protein concentration using three different assays.
3.0 Theory
In this experiment, bovine serum albumin (BSA) and gelatine will be used to act as protein
sample. BSA usually use as a nutrient cell and microbial culture. Besides that, it used to
stabilize some enzyme during DNA digestion and prevention of enzyme adhesion. While, for
gelatine, there are derived from various animal product. Gelatine is common used in food,
cosmetic and pharmaceuticals products. Purified collagen is converted into gelatine by
extraction from raw material at appropriate temperature. Acidic extraction conditions are
extensively used in the industry but the degree of acid varies with different processes

Figure 3.1 Bovine serum albumin structure

Figure 3.2 Gelatine structure

The Lowry method is routinely used for determining the protein content of biological
samples. In this method, protein is first treated with alkaline copper sulfate in the presence of
tartrate, followed by the addition of folinphenol reagent. Tyrosine and tryptophan residues of
protein cause a reduction of the phosphotungstate components of folinciocaulteau reagent to
give a bluish product that contributes toward enhancing the sensitivity of this method. It is 10
times more sensitive than the biuret reaction (Kumar et al., 2005).
The Bradford assay is highly time-sensitive, with precipitation of protein occurring
about 10 min after contact. It is fairly accurate and samples that are out of range can be
retested within minutes. The Bradford assay has become the colorimetric method of choice,
owing principally to its high sensitivity, perceived linearity, and the speed of analysis (Sapan
et al., 1999). The Bradford is recommended for general use, especially for determining protein
content of cell fractions and assessing protein concentrations for gel electrophoresis. The
Bradford assay relies on interactions between basic amino acids residues (primarily arginine,
lysine and histidine) with the Coomassie brilliant blue G-250 dye (CBB) in an acidic matrix.
Both hydrophobic and ionic interactions stabilize the anionic form of the dye, causing a
visible colour change. The assay is useful since the extinction coefficient of a dye-albumin
complex solution is constant over a 10-fold concentration range.
4.0 Procedures
A) Preparation of sample (Bovine)
1. Weighted about 100mg of bovine powder using electronic beam balance.
2. Then put into 1L conical flask to dilute with distilled water.
3. This dilution will produce 100ppm of solution.
4. Then these dilutions diluted to six different dilutions with are 0ppm, 5ppm, 10ppm, 15ppm,
20ppm and 25ppm in six different 250ml conical flask.

5. For each dilution then will divide to three different parts in the test tubes for analysis
process later.
6. Each test tube will contribute 30ml for each concentration.
B) Preparation of sample (Gelatine)
1. Weighted about 100mg of gelatine powder using electronic beam balance.
2. Then put into 1L conical flask to dilute with distilled water.
3. This dilution will produce 100ppm of solution.
4. Then these dilutions diluted to six different dilutions with are 0ppm, 5ppm, 10ppm, 15ppm,
20ppm and 25ppm in six different 250ml conical flask.
5. For each dilution then will divide to three different parts in the test tubes for analysis
process later.
6. Each test tube will contribute 30ml for each concentration.
C) Preparation Lowry reagent
1. Mix one volume of 0.5% copper sulphate pentahydrate, 1% sodium tartrate, with 50%
volumes of 2% sodium carbonate and 0.4% NaOH (Reagent 1)
2. Then diluted commercial Folin-ciocalteu phenol reagent with an equal volume of water
(Reagent 2)
D) Preparation Bradford reagent
1. Dissolved 100mg Coomassie Blue G-25 in 50ml of 95% ethanol.
2. Then, add100ml of 85% phosphoric acid and diluted to one liter volume of distilled water

3. The reagent must be filtered since to precipitate.

E) Analysis protein
(i) Using lowry reagent
1. Mixed 0.25ml of protein with 2.5ml of lowry reagent 1.
2. After 10 minutes, add 0.25ml of lowry reagent 2 and mixed well.
3. Then, after 30 minutes, measure the absorbance at 750nm.
(ii) Using Bradford reagent
1. Mixed 0.25ml of sample with 2.5ml of Bradford reagent.
2. After 5 minutes, measure absorbance at 595nm.
5.0 Apparatus
Apparatus

Beakers
spectrophotometer
Electronic beam balance
Measuring cylinder
Pipette and micropipette
Conical flask (1000ml & 250ml)
Test tubes

Material

Bovine serum albumin (BSA)

Gelatine
10% NaOH
0.3% copper sulphate pentahydrate
1.2% sodium potassium tartrate
Potassium iodide
0.5% copper sulphate pentahydrate
1% sodium tartrate

2% sodium carbonate
0.4M NaOH
Folin-Ciocalteu phenol reagent
Commassie Blue G-250
95% ethanol
85% phosphoric acid

6.0 Results
A) Lowry reagent
Absorbance range (nm)
Concentration (ppm)
0
5
10
15
20
25

Bovine
0.040
0.032
0.045
0.057
0.065
0.073
Table 6.1 Absorbance range using lowry reagent

Gelatine
0.040
0.036
0.039
0.041
0.040
0.047

Absorbance range for lowry reagent

0.08
0.07
0.06
0.05

Absorbance range (nm)

Bovine

0.04

Gelatine

0.03
0.02
0.01
0

10

15

20

25

Concentration (ppm)

Graph 6.2 Absorbance range using lowry reagent

B) Bradford reagent

Absorbance range (nm)

Concentration (ppm)
0
5
10
15
20
25

Bovine
0.357
0..446
0.491
0.498
0.536
0.590
Table 6.3 Absorbance range for bradford reagent

Gelatine
0.357
0.395
0.370
0.364
0.347
0.346

Absorbance range for bradford reagent

0.7
0.6
0.5
0.4

Bovine

Absorbance range (nm) 0.3

Gelatine

0.2
0.1
0

10

15

20

Concentration(ppm)

Table 6.4 Absorbance range for bradford reagent

C) Standard curve (280nm)
Absorbance range (nm)
Concentration (ppm)
0
5
10
15
20
25

Bovine
0
0.005
0.013
0.018
0.023
0.025
Table 6.5 Absorbance range for standard curve

Gelatine
0
0.001
0.004
0.008
0.010
0.021

Absorbance range for standard curve

0.03
0.02
0.02

Absorbance range (nm)

Bovine
Gelatine

0.01
0.01
0

10

15

20

25

Concentration (ppm)

Table 6.6 Absorbance range for standard curve

7.0 Sample Calculations
A) Preparation of bovine and gelatine sample
5ppm in 250ml
M1V1 = M2V2
(100) V1 = (5) (250)
V1 = 12.5ml
10ppm in 250ml
M1V1 = M2V2
(100) V1 = (10) (250)
V1 = 25ml

15ppm in 250ml
M1V1 = M2V2
(100) V1 = (15) (250)
V1 = 37.5ml
20ppm in 250ml
M1V1 = M2V2
(100) V1 = (20) (250)
V1 = 50ml
25ppm in 250ml
M1V1 = M2V2
(100) V1 = (25) (250)
V1 = 62.5ml
8.0 Discussions
To achieve the objective of the experiment, with is to determine protein concentration using
three different assays, this experiment was conducted. This experiment also will give the best
assay for protein estimation. Sample of protein that will use to detect the protein are bovine
serum albumin (BSA) and gelatine. Each sample of protein will through the process of
dilution at six different concentrations with are 0ppm, 5ppm, 10ppm, 15ppm, 20ppm and
25ppm. Then, each dilution will separate into three different test tubes to analysis section.

For lowry reagent analysis, all the sample are changed from yellow to colourless
colour. The absorbance for different concentrations is analysed at 750nm. From Graph 6.2,
bovine and gelatine sample of the absorbance range will increase due to the increasing of
concentration. From the result end of this experiment, the lowry reagent show the sensitivity
for protein estimation. This reagent can detect small amount of absorbance range from both
sample. The absorbance ranges for all concentration are below 0.1 nm. Based on the
generality the graph obtained shows the consistency of the result for both proteins. Besides
that, lowry reagent is very sensitive to the tyrosine present in the protein. But for the linearity,
both samples that are detected by lowry reagent, not give the straight line based on Graph 6.2.
Then, for Bradford reagent, the colours are change from colourless to blue. Graph that
collected from the analysis using Bradford reagent are plotted at the end of experiment. The
absorbance for different concentrations is analysed at 595nm. From Graph 6.4, the absorbance
range of bovine is increase due to increasing of concentration, while, gelatine sample only
increase at concentration 5ppm but then decrease due to increasing of concentration. The
sensitivity of this reagent not high as lowry reagent with is only can detect the protein
absorbance range for both sample below 1.0nm. For generality of Bradford reagent,
absorbance range for bovine sample is consistent increase compare to gelatine. Then, for
linearity of the graph, both samples not give straight line.
Lastly, for standard curve, the concentration is analysed at 280nm. Standard curve is
the best reagent that convenience compares to lowry and Bradford reagent. Standard curve is
the easy reagent to get the reading of absorbance range. From the Graph 6.6, the absorbance
range for bovine and gelatine increase by the increasing of sample concentration. Based on
this graph, the linearity for both protein samples are not in straight line. The increasing of
absorbance range is in roughly due to different concentration of protein in each dilution. For

generality, both protein samples are consistent in increasing of absorbance range while for
sensitivity, standard curve is the best reagent compare to lowry and Bradford reagent.
9.0 Conclusions
This experiment is carried out to reveals the best method on protein estimation by using
three types of assay with are lowry reagent, Bradford reagent and standard curve. The
experiment is done to quantify the estimation of protein (BSA and gelatine) at 0, 5, 10, 15, 20
and 25 ppm concentration.
10.0

Recommendations

1) Weight the protein sample at correct mass for dilution

2) Make correct calculation for sample dilution.
3) See the correct measurement correctly
11.0 References
Fornes O., Garcia-Garcia J., Bonet ., Oliva B. (2014)

Chapter Four On the Use of Knowledge-Based Potentials for the

Evaluation of Models of ProteinProtein, ProteinDNA, and Protein
RNA Interactions

Kumar R., Shukla A. K., Bagga E., Kumari S., Bajpai R. P., Lalit M. Bharadwaj (2005)
1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide interferencewith Lowry method
Sapan, C.V., Lundblad, R.L., Price, N.C., (1999).
Colorimetric protein assay techniques. Biotechnol. Appl. Biochem. 29, 99e108