DEPARTMENT OF BIOLOGY

FACULTY OF SCIENCE & MATHEMATICS

UNIVERSITI PENDIDIKAN SULTAN IDRIS

SBL 1023
LABORATORY TECHNIQUE
IN BIOCHEMISTRY

STUDENT’S NAME NAZIRA BINTI NASLAMROS

LECTURER’S NAME ASSOC.PROF SHAKINAZ DESA
MATRIC NO E20161013757
DATE 29th NOVEMBWE 2017
LABORATORY MP 13
Introduction

Protein are the most important of all biological compounds. The word “protein” is derived

from the Greek “proteios”, that meaning “ of first importance”. Protein is performed a variety

function, including the role ( structure, catalyst, movement, transport, hormones, protection,

storage and regulation ). The properties of protein is it has peptide backbone. Proteins are the

most important nutrients in our body. In this experiment, the amount of protein had been

compared between chicken egg and quail egg. Protein help to catalyse the reaction in our

body.

The biuret reagent detect the presence of peptide bond in shortest time. The biuret reagent

cannot determine the specific concentration. Gelatine is water soluble protein, it derived from

the extraction of collagen that had found in animal skin, bones and tendons. Gelatine is not

found in plant sources.

Objective :

I. To use spectrophotometer correctly

II. To analyse the amount of protein in sample

III. To compare the amount of protein in two different sample.

Procedure :
1. Nine test tube was prepared.

2. Test tube 1 to 7 can be made until two set. This step for increase the precision of the

result.

3. The test tube was vortex to ensure the sample has mixed.

4. The spectrophotometer was switch on and set the wavelength in 550 nm.

5. The small amount of solution was transferred from tube 1 into curvette and the

absorbance was set to zero.

6. The other standard and sample was put inside the spectrophotometer and the

absorbance was determine in different value.

Result :

Test Gelatine Water Concentration Biuret absorbance concentration

Tube (ml) (ml) protein reagent

1 0.0 1.0 0 0.000

2 0.1 0.9 1 0.079

3 0.2 0.8 2 0.144

Add
2 ml of
4 0.3 0.7 3 biuret 0.192
and
5 0.4 0.6 4 incubate 0.276
for 15
minutes
6 0.5 0.5 5 0.317

7 0.6 0.4 6 0.396

8 Sample A ( 1 ml ) 0.254

9 Sample B ( 1 ml ) 0.463
Graph

absorbance
0.45

0.4

0.35

0.3

0.25

0.2

0.15

0.1

0.05

0
0 1 2 3 4 5 6 7

Graph absorbance versus concentration

x-axis = concentration

y-axis = absorbance

orange line = sample A

blue line = sample B

Discussion :

The subunit which make protein are amino acid. The amino acid are joined together

by dehydration synthesis to form chains, which are hundreds of amino acids long which is

called protein. Protein function as enzymes or as structural units in cells. They do most of the

work in cell. They do most of the work in cell. Almost all of exciting stuff such as

metabolism, memory, hormone action, and movement involves in proteins. In this lab, we

have learnt method of measuring concentration of protein, biuret assay.

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 acid to form proteins) and change colour where it does so. The

spectrophotometer has been used to measure the intensity of colour change produced. The

more protein presence the darker the colour.

In order to quantitatively determine how much protein is represented by a particular

absorbance reading it is necessary to construct a standard curve. This is done by performing

the biuret reaction on a species of prepared the solution of gelatine at (0,1,2,3,4,5,6 and 7

mg/ml) in water. The absorbance reading obtained from these solutions are used to construct

a graph of absorbance as function of protein concentration. This graph is called the standard

curve for assay, and can be used to convert the absorbance reading for the experimental

samples (eggs from chicken and quail) into a protein amount or concentration.

Based on the graph that has been constructed, it shows that the standard curve

concentration of protein for the sample as shown in the result. From the graph we can see

that, the highest protein is (quail egg) and the lowest protein is (chicken egg). The sample A

is chicken egg and the sample B is quail egg.

 So from the experiment maybe there are some mistake happen while conduct it.

That’s why our group get the opposite result than the actual one. Error that occur could be the

mixing of using the same pipette.

Conclusion :

We can conclude that there are two ways of measuring the protein concentration in a

solution. It is using the biuret assay. Both can be used to measure protein concentration in a

solution but the most accurate to measure the protein concentration by using lowry assay.

This is because lowry assay can measure the protein as low as 1µ𝑔/𝑚𝑙 to 1500 µg/ml

compared to biuret which can only measured the protein from 5-160µg/ml.

In the experiment we have test two different types of egg (chicken and quail). From

the graph quail egg contain more protein while chicken egg contain the least.

Reference :

I. http://www.cultek.com/otros/perfil-proveedors/pierces-manual-protein-assay.pdf.

II. http://www.Biochemistrygirls.blogspot.my/2013/04/experiment-2-protein-experiment.