SBL1023

TECHNIQUES IN BIOLOGICAL AND

BIOCHEMISTRY LABORATORY

SPECTROPHOTOMETRY (PROTEIN ANALYSIS)

PROTEIN ANALYSIS

NAME: FARAH WAHIDAH BINTI JEPPRI

MATRICS NUMBER: E20161013726
GROUP: A
LECTURERS NAME: ASSOCIATE PROF DR. SHAKINAZ BINTI DESA
 LAB 3 SPECTROPHOTOMETRY (PROTEIN ANALYSIS)
PROTEIN ANALYSIS

INTRODUCTION
Every foods surround us consists of several classes. Most of the foods contain protein
and the amount of protein may different for different types of foods. There are many ways to
measure protein concentration. In this laboratory experiment, the protein was measured by
measuring the absorbance of the coloured product formed by the mixture of protein and organic
molecule. Biuret reagent are the reagent that detect the presence of peptide bonds. The food
extraction was taken and mixed with distilled water and Biuret reagent, so that absorbance can
be taken when the all mixture are placed to be measured. Different methods to yield the protein
concentration have different results depending on the many factors include specificity,
sensitivity, the measurable range of concentration, the accuracy, the nature of the protein to be
examined, the presence of materials interfering with the measurement and the time required for
the measurement. There have their own advantages and disadvantages. The absorbance reading
will reflect the protein concentration, thus we can know and make comparison for different
types of foods have different concentration of protein based on the graph plotted. The protein
intensity affects the intensity of the colour of protein extraction with Biuret reagent, where the
colour will be more intense with more protein.

OBJECTIVES
To determine the protein concentration of solution using the Biuret method of protein
assay.
Learn how to use spectrophotometer in measuring different absorbance of different
protein concentrations.

METHADOLOGY
A) Protein preparation
1. 9 sets of test tubes and prepare gelatin and distilled water was prepared for each test
according to the volume listed below :
Figure 1.0

Test tube Gelatin, mL H2O, mL

1 0 1
2 0.1 0.9
3 0.2 0.8
4 0.3 0.7
5 0.4 0.6
6 0.5 0.5
7 0.6 0.4
8 1 mL of sample A
9 1 mL of sample B
 2. All amount of volume listed was prepared by using micropipette to get the exact
volume.
3. Test tube 8 and 9 was prepared to put the protein samples. 1 ml of protein samples was
carefully pipetted into each test tube.
4. 2 ml of Biuret reagent was added to every test tubes.
5. Each test tubes was incubated for 15 minutes.
6. 1 ml of solution from test tube 1 was transferred into a cuvette and the cuvette was
gently wiped with a paper towel to remove fingerprints and dust.
7. The absorbance was set to zero. This tube will serve as blank.
8. The absorbance of other standards and sample protein of test tube 2 to test tube 9 was
measured using the step as in (1). *DO NOT blank the instrument again.
9. The absorbance of each standards and samples was recorded.
10. The graph of standard curve was plotted using the absorbance value of protein standards
and the absorbance values of protein samples was interpolated.

ATTACHMENT:

The diagram 2.0 shows that solution for every test tube after added with biuret reagent

The diagram 3.0 shows that 1 mL solution of every test tube are transferred into cuvette to
measure the absorbance by using spectrophotometer. From left is taken form test tube 1 and
so on.
RESULTS

Test tube Gelatin H2O, mL Protein 2 mL of 15 minutes Absorbance

conc, Biuret incubation
mg/mL
1 0 1 0 0.000
(BLANK)
2 0.1 0.9 1 0.049
3 0.2 0.8 2 0.104
4 0.3 0.7 3 0.161
5 0.4 0.6 4 0.286
6 0.5 0.5 5 0.347
7 0.6 0.4 6 0.415
8 1 mL of sample A X2 0.209
9 1 mL of sample B X1 0.052

Figure 5.0

X1 X2

Figure 6.0

Value of X2 = 3.40 mg/mL

Value of X1 = 1.30 mg/mL
DISCUSSION

In this experiment, the absorbance of nine test tubes consist of different concentrations
of protein were measured and observed. In this experiment, two method was used to observe
the protein concentrations which are biuret reagent and spectrophotometer. The biuret reaction
is a method that can be used to determine the amount of soluble protein in a solution. The biuret
reagent (copper sulphate in a strong base) reacts with peptide bonds (which join amino acids
to form proteins) and changes colour when it does so. The more protein present the darker the
colour. The spectrophotometer has been used to measure the absorbance of the protein based
on the intensity of the colour produced.
For test tube 1 until 7, the protein concentration already stated and known which is
increasing from test tube 1 until 7 and we just need to measure the absorbance, thus we can
make the relationship between the protein concentration and the absorbance. Instead of
measuring the absorbance of all liquid protein sample, the focus in this experiment also we
want to find the unknown protein concentration of two liquid sample. In this experiment, we
used full cream milk for sample A and chocolate milk for sample B which both types of milk
are from the same brand. Based on the nutritional information, full cream milk contain 6.4g of
protein per serving 200 mL while chocolate milk contain 5.6g of protein per serving 200mL.
The sample were too concentrated and the absorbance cannot be measured by
spectrophotometer. So, we just use 10L of both sample and we need to dilute it by adding 5
mL of distilled water to both test tube. After mixing them, the biuret reagent was added to the
both test tube and including test tube 1 until 7 and we could observe the intensity of colour
produced indicate the amount of protein in the sample roughly.
All sample, test tube 1 until 9 was tested in the spectrophotometer to get the value of
absorbance. We could observe the absorbance reading increase relatively from test tube 1 until
7. From the observation, we can conclude that the higher the concentration of protein in the
sample tested, the higher the absorbance reading. Another two test tube contain sample A and
B were tested and different absorbance observed. Sample A showed 0.209 for it absorbance
and 0.052 for sample B. Sample A have higher absorbance compared to sample B which
indicate that sample A have higher concentration of protein compared to sample B. The graph
was plotted from the data collected and we extrapolated the absorbance of both sample A and
B to get their protein concentration. From the graph, the protein concentration of sample A was
3.40 mg/mL which higher than sample B, 1.30 mg/mL. When we made comparison from the
nutritional information stated at the milk packing, it was clearly stated that sample A have
higher mass of protein per serving 200 mL which 6.4 g and 5.6 g per serving 200 mL. The
result we got satisfied the information that full cream milk contain higher amount and
concentration of protein compared to chocolate milk. The experiment was successfully carried
out.
REFERENCES
1. Archived on 5 December 2017 from
https://www.scribd.com/document/366288819/sbl-report3-
protein#download&from_embed

2. Lecture note, Protein(The Analysis)

3. Laboratory Manual SBL 1023
4.