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Protein analysis and concentration


The human body uses proteins for many things, including repairing and building tissues,

acting as enzymes, aiding the immune system, and serving as hormones. Each of these important

functions requires a slightly different form of protein. In spite of their differences in structure, all

proteins contain the same basic sub-components.

Earlier, we mentioned that protein plays a role in tissue repair, and that's why it's so

important to have protein in your diet. But what are the best sources of protein? Afterwards, you

might take a look in your refrigerator and decide whether your diet is protein rich or protein


1. To observe the colour of solution depending on its different concentration of protein

2. To determine protein concentration using protein assays which is Biuret assay

3. To determine unknown concentration of different protein samples

4. To learn how to use spectrophometer in measuring the absorbance of different protein



 Stock solution of protein

 Deionized water (dH2O)

 Test tubes and tube rack

 Pipette

 Biuret reagent

 Spectrophometer

 Protein samples (Pony Fish “ikan kekek” & Torpedo Scad “ikan cencaru”)

a. Preparation of biuret reagent

1. We prepared the biuret solution, by adding, 300ml of 10% (w/v) NaOH to 500ml of a

solution containing 0.3% copper sulphate pentahydrate and 1.2 sodium potassium

tartarate the dilute to 1 liter.

b. Protein preparation

We prepared the test tube as shown in the table below

1. First we labelled the test tube using sticker paper to make easier for us to record the


2. We added different amount of gelatine in each tube.

3. Then we also pipetted the different amount of water in each tube to make sure the

concentration diluted.

4. The we pipetted the biuret reagent in each test tube

Test tube Gelatin (ml) H2O (ml) Biuret Incubation

1 0 1 2ml

2 0.1 0.9 2ml

3 0.2 0.8 2ml

15 minutes incubation
4 0.3 0.7 2ml

5 0.4 0.6 2ml

6 0.5 0.5 2ml

7 0.6 0.4 2ml

8 1ml of sample A 2ml

9 1ml of sample B 2ml

c. Determine protein concentration

1. 1ml of solution from tube 1 was transferred into a cuvette and wiped the cuvette with

a paper towel to remove fingerprints and dust.

2. The absorbance set to 'zero'. The tube served as 'blank'.

3. The absorbance of the other standards and sample proteins were measured using the

step as in (1).

4. The absorbance were recorded of each standards and samples.

5. A graph was plotted of standard curve using the absorbance value of protein

standards and interpolate the absorbance values of the protein samples


Test tube Absorbance Protein concentration

1 0.000 0

2 0.036 1

3 0.085 2

4 0.088 3

5 0.122 4

6 0.291 5

7 0.297 6

8 (Torpedo Scad) 0.195 7

9 (Pony fish) 0.064 8


In this experiment, we would like to analyse the absorbance of protein concentration

prepared. We used gelatine as our source of the protein but with different concentration in each

tube. Gelatine is yellowish, odourless and nearly tasteless substances that are made by prolonged

boiling of skin, cartilage, and bones from animals. It is a mixture of peptides and proteins

produced by partial hydrolysis of collagen extracted from skin, bones, and connective tissues of

animals such as domesticated cattle, chicken, and fish.

From tube 1 to tube 7, each tube were added 0.1 ml until 0.6 ml of gelatine in each tube.

Tube 1 is a negative control which contains no gelatine at all with 1 ml and 2ml of biuret in it. As

the result, the absorbance shows 0.00. The absorbance increased proportionally with the

increasing of the protein concentration in each tube. Test tube 7 shows the highest absorbance

0.297 with the 60% gelatine in it.

Me and my laboratory partner decided to bring our laboratory samples that is Torpedo

Scad and Pony fish.

Torpedo Scad fish also known as Cordyla Scad, Finny Scad, Hardtail Scad, and Horse

Mackerel. It is found in large schooling fish in Indo west Pacific.

Torpedo Scad Pony Fish

Ponyfishes are small and laterally compressed in shape, with a bland, silvery colouration.

They are distinguished by highly extensible mouths, and the presence of a mechanism for

locking the spines in the dorsal and anal fins. We would like to determine which fish have the

highest portion of protein. First of all, we separated the bones from the fish and blended their

fillet to make sure that it is easier for us to extract their protein. After that, for about 0.2 g of fillet

were separated and were crushed with mortar and pestle. Then, diluted with water for about 10ml

to 12 ml depends on how the concentration look. If it too concentrated, then add more.

The solution of the fillet then undergoing a process which is to separate the fluid from the

fillet . We only want the protein extracted from the fillet in a liquid form. Vortex and centrifuge

it for about few minutes to get the protein extraction. When the liquid is ready proceed the

procedures stated.

We does not know the protein concentration from the protein extraction. But we do have

their absorbance value. So , we can plot a graph of absorbance versus protein concentration of
the 7 tubes prepared before using gelatine. The absorbance for pony fish and torpedo scad

protein must be in between range from test tube 1 to test tube 7. So we get the value 4.2 for the

torpedo scad which contains higher protein concentration rather than pony fish protein with the

value of 1.3.


1. Nutrition source


2. Protein analysis techniques


It is actually new thing for most of my classmates as it is our very first time using and

experiencing to use spectrophotometer. Our lecturer Dr. Shakinaz brief to us about how to use

and how spectrophotometer actually works. Learn from mistakes is a must. In order to be good in

laboratory, we must apply those step and if we do make some mistake, learn from it. I do. We all

do actually. The spectrophotometer cannot read our protein sample as the sample is too

concentrated. We need to dilute the protein sample so that the spectrophotometer can read ours.

During our laboratory session, at first the spectrophotometer cannot read our protein sample.

After another preparation of protein sample with a different dilution techniques, we manage to

record it reading.