RESULTS AND DISCUSSION

Data Collection and Observation

Table 1: The absorbance values at different biomass concentrations

Biomass
0
0.001
0.0005
0.0002
0.000125
Concentration
5
(g/ml)
Absorbance Value
0
0.754
0.623
0.585
0.472
(A)
Table 2: Biomass Concentration for a given culture suspension
Absorbance Value (A)

0.392

Biomass Concentration (g/ml)

2.02 x 10-4

0.0000625

0.328

Absorbance Value vs Protein Concentration

0.8

f(x) = 565.35x + 0.28

R = 0.63

0.7
0.6
0.5

Absorbance Value (A) 0.4

0.3
0.2
0.1
0

Protein Concentration (mg/ml)

Figure 1: Graph of Absorbance value vs Biomass Concentration

Based on the data recorded in Table 1, it shows that when the biomass concentration
increases, the absorbance values also increases. The value for the unknown biomass
concentration is determined from the calibration curve plotted. The OD reading is the y-

intercept of the linear graph and the value is submitted into the y = mx + c to obtain x value
(biomass concentration).

Data Analysis and Discussion

In

this

experiment,

dilution

of

Bovine

Serum

Albumin

(BSA)/Immunoglobulin (IgG) with sodium chloride (NaCl) with different

biomass concentration (range from 0 - 0.0000625g/ml) is tested using UV
spectrophotometer to obtain the protein concentration of this stock
suspension as 0.01 g/ml. The UV spectrometer measures the amount of
light that is absorbed by the sample in the spectrometer at 595nm. Before
taking the OD readings, each samples is added 1ml of Bradford Protein
reagent and incubated for 10 minutes. The Bradford protein assay is used
to measure the concentration of total protein in a sample. The principle of
this assay is that the binding of protein molecules to Coomassie dye under
acidic conditions results in a colour change from brown to blue. This
method actually measures the presence of the basic amino acid residues,
arginine, lysine and histidine, which contributes to formation of the
protein-dye complex.
The purpose of the incubating the mixture sample are because color formation of the
Bradford reagent is continues to occur after the incubation period as the Bradford reagent do
not have a stable endpoint and. The incubation step is also needed as it allows the color
reagent to bind to proteins with a subsequent shift in the reagents absorbance spectrum. The
samples need to be incubated in order to let the mixture of BSA, NaCl and Bradford reagent
react properly.

The result obtained can be seen in the Table 1 and it is following the
theory. The absorbance values is increases as the biomass concentration
is increases. The calibration curve of absorbance values versus biomass
concentration is shown in the Figure 1. From the graph, it shows that the
absorbance values is directly proportional to the biomass concentration.
This is because when the higher concentration of solution is used, the
spectrophotometer absorbs more light as the light is not able to pass
through the solution totally.
Furthermore, from Table 2, it shows the calculated value of biomass
concentration for a given unknown sample. During the experiment, a
sample of unknown biomass concentration is provided and the biomass
concentration must be determined. In order to calculate its biomass
concentration, the OD reading of the sample is determined first which is
0.392 A. From the OD reading, biomass concentration is determined from
the calibration curve and the calculation is as follows
y = mx + c where y = 0.392
y = 565.35x + 0.2778
0.392 = 565.35x + 0.2778
x = 2.02 x 10-4 g/ml
The biomass concentration of the sample obtained is 2.02 x 10-4 g/ml which is
deviated to the theoretical value at 0.01 g/ml. The differences between trend lines in
experimental data may due to some errors that occur during the experiment such as the
BSA/IgG dry and NaCl solution might not mixed well and form sediment at the bottom of the

cuvette. However, the error in the result obtained is expected as the calibration curve is not
100% accurate as theoretical calibration curve. The fingerprint at the cuvette also will affect
the result as the light cannot pass through the yeast as it is blocked.
An important feature of a colorimetric protein reagent is that it should have a stable
endpoint, thus allowing color formation to reach a plateau. The samples also should be
incubated at room temperature for no more than 1 hour as the absorbance will increase over
time.