EXPERIMENT 1 : PROTEIN ASSAY
OBJECTIVES:
1. To compare the protein concentration of Tilapia Merah (Oreochromis niloticus) for freshwater and Kayu Hitam (Euthynnus affinis) fish for the marine water.
2. To differentiate the protein concentration of both fishes.
APPARATUS, MATERIALS AND METHOD:
1. To compare the protein concentration of Tilapia Merah (Oreochromis niloticus) for freshwater and Kayu Hitam (Euthynnus affinis) fish for the marine water.
2. To differentiate the protein concentration of both fishes.
APPARATUS, MATERIALS AND METHOD:
- The solution of gelatines at 1, 2, 3, 4, 5 and 6 mg/mL of Biuret assay are prepared in water to get it standard of protein concentration.
- For dilution, the volume of gelatines are measured to show the different concentration of gelatines before. The formula used is M1V1=M2V2.
- Then, 1.7mL of distilled water is added for dilution to form 5 mg/mL of gelatines. This calculation is applied to prepare the rest different concentration of gelatines.
- 10mL measuring cylinder, beakers and dropper are used to prepare the solutions.
- 0.5mL of all the proteins are measured with different concentration and Biuret reagent is added for each proteins. The colour changes are observed.
- Before measure the absorbance of each protein, left the protein in 10 minutes.
- The absorbance of the standards of the samples, is measured by using a spectrophotometer. A spectrophotometer consists of a spectrometer to produce light of a specific colour and a photometer to measure the intensity of the light. Here, the wavelength for Biuret assay is 540nm while for Lowry assay is 750nm.
- Then, 1mL of each gelatine with different concentration is put into the spectrophotometer to measure their absorbance to plot the standard curve. The standard curve is used to determine the concentration of the protein in the fishes.
- The steps are repeated to prepare the solution of gelatines at 0.1, 0.2, 0.3, 0.4, 0.5 and 0.6 mg/mL in water of Lowry assay to plot the standard curve.
- Two standard curve graphs are plotted.
- Next, for Biuret method, the samples is filtered and centrifuged at 4 degree celcius, 14000rpm in 15 minutes.
- The supernatant then is collected. After that, the samples are filtered again using Whatman filter No 1.
- Lastly, the protein content is measured against gelatine standard using Lowry or Bradford method.
CONCLUSION:
As the conclusion, we can observed that the concentration of protein in Tilapia Merah (Oreochromis niloticus) for freshwater is higher compared to Kayu Hitam (Euthynnus affinis) fish for the marine water. Based on Biuret method result, the absorbance for Euthynnus affinis fish is 0.399A while in Lowry method for Oreochromis niloticus fish, its absorbance is 0.975A. Otherwise, based on Biuret equation that given which is y=0.1276ln(x)+0.0346 it proved that the protein concentration of Euthynnus affinis fish is 17.3993A while the protein concentration of Oreochromis niloticus fish with Lowry equation is y=0.0405x-0.015 is 24.444A.
From this results, we can conclude that the environment of the fish also important and could influence the protein concentration of the fish. As we know, the concentration of freshwater are lower that the marine water so that the muscle of the fish will only lost little amount of water. Therefore, their protein concentration are higher compared to marine fishes
As the conclusion, we can observed that the concentration of protein in Tilapia Merah (Oreochromis niloticus) for freshwater is higher compared to Kayu Hitam (Euthynnus affinis) fish for the marine water. Based on Biuret method result, the absorbance for Euthynnus affinis fish is 0.399A while in Lowry method for Oreochromis niloticus fish, its absorbance is 0.975A. Otherwise, based on Biuret equation that given which is y=0.1276ln(x)+0.0346 it proved that the protein concentration of Euthynnus affinis fish is 17.3993A while the protein concentration of Oreochromis niloticus fish with Lowry equation is y=0.0405x-0.015 is 24.444A.
From this results, we can conclude that the environment of the fish also important and could influence the protein concentration of the fish. As we know, the concentration of freshwater are lower that the marine water so that the muscle of the fish will only lost little amount of water. Therefore, their protein concentration are higher compared to marine fishes