SBL 1023

Technique in Biology And

Biochemistry Laboratory

Lab 3: Spectrophotometry And

Protein Concentration Measurement

AYU ILYANA BT ZULKIFLI

STUDENT`S NAME
( E20161014077)

GROUP B

Lab 3: Spectrophotometry and protein

EXPERIMENT`S NO
concentration measurement

DATE/DAY/PLACE 21/11/2017 /TUESDAY/ B2-L3-MP 14

LECTURE`S NAME Professor Madya Dr. Shakinaz Binti Desa

Lab 3: Spectrophotometry and protein concentration measurement

1.0 INTRODUCTION
In this lab, we investigate the present of protein concentration in a cucumber by using
2 method that is biuret test and spectrophotometry test. We wonder if there has any present
of protein in a cucumber as we know in a cucumber, there are high in vitamin K, vitamin C,
manganese and others nutrition. So, we decided to make an experiment on the cucumber.
We made observation on 2 sample that is the cucumber peel and it cucumber flesh. We
want to know which one contained more higher in protein. The cucumber peel or cucumber
flesh?
First of all, the sample was cut into small pieces and was grained, this is to extract
the protein. Then, to determine the presence of peptide bond in the cucumber, we use biuret
protein reagent. When the peptide bonds present in an alkaline solution, the coordination
complexes associated with a copper ion (Cu2+) are violet in colour. Nitrogen atoms of the
peptide bonds form a coordination bond with metal ion. The quantity of the complexes
formed is proportional to the number of peptide bonds. Thus protein intensity affects the
intensity of the colour, where the colour will be more intense with more protein.
Then, to determine the concentration of a substance in a solution, we used
spectrophotometry. We determined the concentration of protein in cucumber sample by
measured the absorbance of the wavelength in the sample with the standard curve. Thus, a
graph of absorbance and concentration of protein was plotted.

2.0 OBJECTIVE
 Learn how to prepare a tissue protein extract and use a protein assay reagent to
measure the protein concentration.
 Theory and practice of absorbance spectrophotometry.
 Learn how to prepare and use a standard curve and absorption spectrum.
 Differentiation protein concentration contained in cucumber peel and cucumber flesh.

3.0 MATERIALS
 (BSA)- Gelatine , 10mg/ml
 Deionized water (dH2O)
 Test tubes and stand
 Pipette
 Biuret reagent
 Spectrophotometer
 Protein samples

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4.0 METHODOLOGY

(A) Protein preparation

1. 1 sets of test tubes with the numbers 1 to 6 and the gelatine stock solution (10 mg/ml) was
prepared according to the concentration listed below:
gelatine
BSA stock
Tube conc. H2O (ml)
(ml)
(mg/ml)
1 0 1.0 0
2 1 0.9 0.1
3 2 0.8 0.2
4 3 0.7 0.3
5 4 0.6 0.4
6 5 0.5 0.5
7 6 0.4 0.6
Table 1.0: preparation of stock solution
2. Duplicate test tubes was prepared for protein samples and 1ml of the protein samples was
carefully pipetted into each tubes.
3. 2ml of biuret reagent was added to every tube, the 7 tubes for the standard curve and the
duplicate tubes for protein samples.
4. The tubes was covered with parafilm and briefly vortex to ensure that the protein samples
and the biuret reagent are thoroughly mixed.
5. Allowed the tubes to stand at 15 minutes.
6. The spectrophotometer was switch on and adjusted the wavelength to 550nm.

(B) Determined protein concentration

1. Transferred 1ml of solution from tube 1 into a cuvette and gently wipe the cuvette with a
paper towel to remove fingerprints and dust.
2. The absorbance was set to 'zero'. This tube will serve as 'blank'.
3. The absorbance of the other standards and sample proteins was measured used the step
as in (1).
4. The absorbance of each standards and sample was recorded.
5. A graph of standard curve was plotted using the absorbance value of protein standards
and interpolate with the concentration of protein sample

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5.0 DATA, GRAPH AND CALCULATION

5.1 DATA
Concentration
Gelatine H2O
Test tube of protein Absorbance Biuret
(mg/ml) (ml)
(mg/ml)
1 0 1.0 0 0
2 0.1 0.9 1 0.180
3 0.2 0.8 2 0.277
4 0.3 0.7 3 0.391
Add 2 ml of
5 0.4 0.6 4 0.526
Biuret.
6 0.5 0.5 5 0.619
Incubate for
7 0.6 0.4 6 0.684
15 minutes
Sample A – Cucumber skin
8 6 0.746
(1 ml)

Sample B – Cucumber flesh 0.342

9 2.6
(1 ml)

Table 2.0. Concentration of test tube 1-6 and concentration of sample A and sample B

5.2 GRAPH

standard curve of absorbance value

0.8 0.746

0.7 Sample A : cucumber peel

uv absorbance : 0.746 0.619
con.of protein : 6 mg/ml
0.6
0.526
Sample B : cucumber flesh
0.5
uv absorbance : 0.342
con.of protein: 2.6 mg/ml 0.391
absorbance

0.4
0.277
0.3
0.18
0.2

0.1
0
0
0 1 2 3 4 5 6 7
concentration of protein (mg/ml)

Graph 1.0: standard curve of absorbance value (mg/ml)

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5.3 CALCULATION

𝒂) 𝑪𝒐𝒏𝒄𝒆𝒏𝒕𝒓𝒂𝒕𝒊𝒐𝒏 𝒐𝒇 𝒔𝒂𝒎𝒑𝒍𝒆 𝑨 𝒂𝒏𝒅 𝒔𝒂𝒎𝒑𝒍𝒆 𝑩, 𝒇𝒓𝒐𝒎 𝒈𝒓𝒂𝒑𝒉

𝑺𝒂𝒎𝒑𝒍𝒆 𝑨 ∶ 𝑾𝒉𝒆𝒏 𝒂𝒃𝒔. = 𝟎. 𝟕𝟒𝟔 , 𝒕𝒉𝒖𝒔 𝒄𝒐𝒏. 𝒐𝒇 𝒑𝒓𝒐𝒕𝒆𝒊𝒏 = 𝟔 𝒎𝒈/𝒎𝒍

𝑺𝒂𝒎𝒑𝒍𝒆 𝑩 ∶ 𝑾𝒉𝒆𝒏 𝒂𝒃𝒔. = 𝟎. 𝟑𝟒𝟐 , 𝒕𝒉𝒖𝒔 𝒄𝒐𝒏. 𝒐𝒇 𝒑𝒓𝒐𝒕𝒆𝒊𝒏 = 𝟐. 𝟔 𝒎𝒈/𝒎𝒍

𝒃) 𝑪𝒂𝒍𝒄𝒖𝒍𝒂𝒕𝒆 𝟏 𝒈 𝒐𝒇 𝒑𝒓𝒐𝒕𝒆𝒊𝒏 𝒇𝒐𝒓 𝒆𝒂𝒄𝒉 𝒔𝒂𝒎𝒑𝒍𝒆

Sample A, cucumber peel Sample B , cucumber flesh

4.55 g of cucumber peel = 6.00 mg/ml of 5.77 g of cucumber flesh = 2.6 mg/ml of
protein protein
1 g of cucumber peel = 1.3186 mg/ml of 1 g of cucumber flesh = 0.4506 mg/ml of
protein protein
Calculation from experimental concentration
2.6 mg/ml x 5 ml =
6.00 mg/ml x 5 ml (volume diluted sample) =
0.013 g of protein in 1 grams of cucumber
0.03 g of protein in 1 grams of cucumber peel.
flesh
In the conclusion, sample A have the high protein than the sample B as its protein
concentration higher by 1 grams of servings gave 0.03 g of protein.

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 6.0 RESULT AND DISCUSSION

6.1 RESULT

Test tube
1-6

Diagram 1.0, from left to right, contain concentration of protein 0, 1,

2,3,4,5, and 6 (mg/ml) in each tube.
Observation Difference in colour
Table 3.0: Observation on test tube 1- 6
Sample A and sample B
Sample Observation
Sample A

Turn dark blue greenish

Sample B

Turn light blue

Table 4.0: Observation on sample A and sample B

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6.2 DISCUSSION
In this lab, we used two method to determine the concentration of protein which are
Biuret test and spectrometry test. Biuret test was used to detect the presence of peptide
bond. It was treated with copper sulphate solution. Biuret test in presence of alkali, protein
reacts with copper (II) ions, (blue in colour) to form a violet coloured complex called biuret.
While, in this lab we used cucumber as a sample, so after we added 1 ml of sample
solution into the test tube contained the biuret reagent, gelatine and distilled water, it show
negative result, no violet colour was present. Instead, the colour was change to dark blue
greenish in colour, due to green pigment on the cucumber. Besides, biuret test was low
sensitivity and that it requires at least 1 mg of protein to be tested. Moreover, the protein
that was contained in a cucumber was very low which about 0.03 g of protein for 1 g of
cucumber peel and 0.013 g of protein for 1 g of cucumber flesh.

Then, spectrophotometry was used to determine the concentration of protein in a

sample solution. It is widely used for detection of proteins or peptides during their separation
by chromatography. From the graph 1.0, sample A which is cucumber peel contain higher
concentration of protein than cucumber flesh, the concentration of protein was difference by
3.4 mg/ml. This is due to different in UV absorbance of the spectrophotometry, which are
cucumber peel, 0.746 light absorbance, while cucumber flesh is 0.342 light absorbance.
When we know the UV absorbance, we can know the concentration of protein from the
graph. In the same cases, the higher the UV absorbance, the higher the concentration of
protein.

Besides, the colour of solution in each test tube also different, in the test tube 6, they
are more intense in colour, it more dark blue in colour than the first test tube. I assume, it
contain more protein than the first one. From the data, test tube 6 contain 0.5 gelatine and
0.5 distilled water with 2 ml biuret reagent. So, I can conclude that due to higher gelatine
contained, the colour was more intense. While, in the test tube 1, the solution was more
diluted, it contain more distilled water than the gelatine. This different is to make a
standard curve to determine the concentration of protein in a sample that was observed
which are in this lab, we observed protein containing in a cucumber. In fact, gelatine is also
a protein, substance derived from collagen, a natural protein present in the tendons,
ligaments and tissues of mammals. It used as a protein concentration standard in lab
experiment.

Back to the graph, we should get a linear graph for standard curve, but the graph
was not in linear, this is because of parallax error of the spectrophotometric.

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 Spectrophotometric measurements are affected by many factors, such as the type of
solvent used, temperature, wavelength of light at which the measurements are made, and
presence of impurities in the cucumber being studied. To eliminate absorbance due to the
solvent, the spectrophotometer must always zeroed (tared) against the solvent. Another
potential problem is light scattering is due to suspended particles. The particulates will
deflect light rays and cause an artificial increase in absorbance. To avoid light scattering, it
is common practice to centrifuge the sample before measuring absorbance to remove
particulates.

7.0 REFLECTION

In 28 November 2017, we had done the spectrophotometry and protein concentration

measurement. This lab include learned how to use the spectrophotometry, and also how to
prepare the protein concentration to get the absorbance value from the spectrophotometry
and lastly how to read the concentration of pH from the graph. Through the graph, we can
know the concentration of pH, with the absorbance value. Lastly, this lab was done to know
whether our specimen has protein or not. In this lab, we use cucumber as our specimen and
the result show that the cucumber has a protein, but in a very small quantity. We determined
the protein by the biuret and the spectrophotometry test. Our result show that, the sample A
which is cucumber peel contained higher protein concentration than the sample B, which are
cucumber flesh. Thus, my lab partner, Dava and I, we highly recommended people to eat
more cucumber peel than the cucumber flesh and don’t just throughout the cucumber peel
when we ate it because there are various nutrient and advantages in a slice of cucumber.

8.0 REFRENCES
1. Marietta, 2017, Measuring Protein Concentration
Through Absorption Spectrophotometry,

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http://w3.marietta.edu/~spilatrs/biol309/labexercises/Spectrophotometry.pdf . Retrieved on
3/12/2017
2. Wikipedia, 3 December 2017, Gelatine, https://en.wikipedia.org/wiki/Gelatin . Retrieved on
3/12/2017