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Group 2

Date performed
Date finished

Exercise 4.2
Determination of Protein Concentration by Spectrophotometry
I.

Introduction
In the laboratory, the importance of determining the
protein concentration is not just limited to knowing the protein
content of a certain sample. This is also done in order to
identify the purity of the protein, as well as a gauge in
knowing how much of the samples should be loaded for tests
of protein purity, identity, and activity. Thus, various ways of
estimating or determining the protein concentrations of
samples have been developed.
Spectrophotometry is one of the most widely used
techniques in protein concentration determination. A
spectrophotometer,
specifically
an
absorbance
spectrophotometer, makes use of the light that is transmitted
through a given solution. Any given object tends to absorb
light, however each one absorbs light at different
wavelengths;
the
same
concept
runs
in
the
spectrophotometer. Thus, it is used to distinguish differences
among various compounds depending on their absorption,
which is also directly related to the concentration of the
absorbing components of the sample. Mathematically, this
relationship between concentration and absorbance is
expressed by the Beer-Lamberts Law (Beers Law), given by
the equation:
A= -log T = log Io/I = bc,
This law states that the amount of light energy absorbed or
transmitted by a solution is an exponential function of the
concentration of absorbing substance present and the sample
length. This equation, almost similar to that of a line function
y=mx+b, yields a straight line called the standard curve. This
curve acts as a calibration in order to properly estimate the
concentration of a certain substance. However, it is not
advisable to extrapolate values beyond the highest
absorbance measured.
The Biuret test or Biuret Assay is a colorimeteric test for
the detection of at least two peptide bonds in a molecule. The
intensity of the purple color of the solution is directly
proportional to the concentration of the protein sample in a
given solution.
At the end of the laboratory exercise, the students should
be able to apply the principles of spectrophotometry in order

to determine and analyze the results

concentration of crude egg albumin.

for the protein

II.

Materials and Methods

A. List of Necessary Chemicals, Glasswares, and Equipment
1) Chemical Reagents
CuSO45H2O
NaKC4H4O6H2O
10% NaOH
0.05 M NaOH
20 mg/mL bovine serum albumin (BSA)
dH2O
2) Glasswares
Pipettes
Stirring rod
Beakers
Test tubes
Graduated cylinder
Erlenmeyer flask
3) Equipment
Vortex mixer
Magnetic stirrer
Analytical balance
Spectrophotometer
B. Schematic Diagram
C. Set-up
D. Sample Calculations

III.

Data Blanks
(page 22 of manual)

IV.

Waste management
Test tubes 1-6: C301
Test tubs 7-11: D499