PROTEIN ASSAY
The difference of protein concentration in raw and fried fish
Objectives:
Procedure:
- To observe either raw fish or cooked fish contain high concentration of protein.
- To learn the use and functions of spectrometer and micropipette.
- Examine the Lowry and Biuret Test to test the presence of protein.
Procedure:
- We prepared the stock of raw fish (ikan merah/ red snapper fish) and cooked fish (fried fish) and left it for about 24 hours to be used on the next day.
- The experiment was conducted in a group of five and each member has their own part to be done. Our group’s leader prepared the standard solutions of gelatin in water for both Lowry and Biuret assay. While the rest of us prepared the raw and cooked fish stock that we have prepared to be centrifuged.
- Both fish stock were filtered using Whatman no 1 filter, filter funnel and volumetric flask to separate the precipitation and supernatant solution. Then about 1.5ml of the supernatant solutions were transferred into centrifuged tube and labelled to be centrifuged in 15 minutes.
- For Biuret assay, 0.5ml protein stock of raw fish was mixed with the 2.5ml of Biuret reagent in the test tube and then incubated for 10 minutes. We use the micropipette to transfer the solutions into separated cuvettes.
- For Lowry assay, 0.25ml protein stock of cooked fish was mixed with the 2.5ml of Lowry reagent 1 and incubated for 10 minutes. After that, 0.25ml of Lowry reagent 2 was added into the solution and incubated for 30 minutes. We use the micropipette to transfer the solutions into separated cuvettes.
- Absorbance for both Biuret (540nm) and Lowry assay (750nm) were measure using spectrophotometer.
- Lastly, the results were compared with the standard measurement of gelatin.
Results:
BIURET ASSAY
Gelatin standard values (Biuret assay) M1V1=M2V2 V1= (1, 2, 3, 4, 5 and 6 mg/ml) (10ml) (6 mg/ml) V1= 1.67 ml, 3.33 ml, 5.00 ml, 6.67 ml, 8.33 ml and 10.00 ml Concentration, x (mg/ml): Absorbance, y (A):
Absorbance of protein (raw fish) = 0.280 A y = 0.1276 ln (x) + 0.0346 ln x = 0.280 – 0.0346 0.1276 x = 6.84 mg/ml (protein concentration)
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LOWRY ASSAY
Gelatin standard values (Lowry assay) M1V1=M2V2 V1= (0.1, 0.2, 0.3, 0.4, 0.5 and 0.6 mg/ml) (10ml) (6 mg/ml) V1= 0.17 ml, 0.33 ml, 0.50 ml, 0.67 ml, 0.83 ml and 0.10 ml Concentration, x (mg/ml): Absorbance, y (A):
Absorbance of protein (cooked fish) = 0.619 A y = 0.0405 x – 0.0151 x = 0.619 + 0.0151 0.0405 x = 15.66 mg/ml (protein concentration)
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Discussions:
From this experiment, we have learnt and replenished our skills in handling equipment and method in the lab sessions such as filtration method, micropipette, centrifuge and spectrophotometer. Filtration method required filter paper (Whatman no 1 filter) and filter funnel as its main apparatus. It is used to separate the stock solutions between the precipitation and the supernatant. Next, the used of micropipette in this experiment was to measure the volume of the stock to be transferred into centrifuge tubes, test tubes and the cuvettes. Micropipette’s reading is more accurate than the pipette, the unit is in microliter (1000µl = 1ml). It can measure from 100 µl until 1000 µl. The tip of the micropipette should be changed every time it’s being used to avoid contamination and reading errors. While for centrifuge laboratory, it was used to separate the liquid of denser particles from the less dense particles. The centripetal acceleration move the denser particles to outwards and less dense particles to the centre. Lastly, spectrophotometer was used to measure the absorbance of the stock solution. Its use photon wavelength that would be absorbed by the solution depends on its concentrations. In this experiment, we use wavelength of 540nm for the Biuret test and 750nm for the Lowry test. Each time we used the spectrophotometer we have to reset with the blank reagent to avoid any errors in the readings.
Apart from that, we have learnt how to plot the graph and make comparison between the concentrations of both raw and cooked fish protein concentration. Before that, the volume of the stock needed should be calculated using its certain formula:
M1V1=M2V2
Where:
M1= Concentration of stock given
V1= Volume of stock needed
M2= Concentration of diluted solution given
V2= Volume of diluted solution given
Where:
M1= Concentration of stock given
V1= Volume of stock needed
M2= Concentration of diluted solution given
V2= Volume of diluted solution given
The concentration of the raw and cooked fish were calculated using its certain formula based on their test because of its absorbance exceed the maximum value of the standard gelatin.
y = mx + c
For Biuret assay:
y = 0.1276 ln (x) + 0.0346
For Lowry assay:
y = 0.0405 x – 0.0151
For Biuret assay:
y = 0.1276 ln (x) + 0.0346
For Lowry assay:
y = 0.0405 x – 0.0151