PROTEIN REFLECTION
Comparison of protein absorbance between marine and freshwater fishes.
OBJECTIVE FOR THIS EXPERIMENT
1) To compare the protein absorbance between marine and freshwater fishes.
2) To apply the Biuret and Lowry method in calculation of protein concentration.
3) To measure the absorbance and compare the concentration using both methods.
2) To apply the Biuret and Lowry method in calculation of protein concentration.
3) To measure the absorbance and compare the concentration using both methods.
STEP BY STEP
1. Two species of fish were choose as sample for this experiment .
2. The fish was weighed at 20g per species of fish using analytical balance.
3. The Phosphate Buffer Saline solution were added into each sample with ratio 1:10 into
beakers.
4. The two mixture of sample and PBS solution were blended to macerate the fish into smaller
size.
5. The 2 sample were placed into conical flask and left for 24 hours at 4C in 100 rpm shaker.
6. Then, the sample were filtered to get the pure stock of sample.
7. The stock were put in sample container by using micropipette.
8. The stock were centrifuged 14,000 rpm with 4C in 15 minutes.
9. The protein content in each sample were measured by using Biuret method or Lowry method.
10. The stock of Bullet tuna were measured by 0.5mL and added with 2.50mL of Biuret reagent.
11.The stock of Oreochromis niloticus were measued by 0.25mL and added by 2.50 Lowry
reagent 1 and incubated within 10 minutes before added by 0.25 Lowry reagent 2.
12. Both solution were put into the test tube shaked to ensure the solution mixed well.
13. The mixture were incubated within 10 minutes.
14. The mixture then pour into the cuvette to measure the absorbance in spectrophotometer.
15. The step 13 was repeated as 5 times to get the average value for the protein absorbance.
16. The protein concentration was calculate using formula y=0.1276 ln(x) + 0.0346 for Biuret
method and y= 0.0405x - 0.015 for Lowry method.
2. The fish was weighed at 20g per species of fish using analytical balance.
3. The Phosphate Buffer Saline solution were added into each sample with ratio 1:10 into
beakers.
4. The two mixture of sample and PBS solution were blended to macerate the fish into smaller
size.
5. The 2 sample were placed into conical flask and left for 24 hours at 4C in 100 rpm shaker.
6. Then, the sample were filtered to get the pure stock of sample.
7. The stock were put in sample container by using micropipette.
8. The stock were centrifuged 14,000 rpm with 4C in 15 minutes.
9. The protein content in each sample were measured by using Biuret method or Lowry method.
10. The stock of Bullet tuna were measured by 0.5mL and added with 2.50mL of Biuret reagent.
11.The stock of Oreochromis niloticus were measued by 0.25mL and added by 2.50 Lowry
reagent 1 and incubated within 10 minutes before added by 0.25 Lowry reagent 2.
12. Both solution were put into the test tube shaked to ensure the solution mixed well.
13. The mixture were incubated within 10 minutes.
14. The mixture then pour into the cuvette to measure the absorbance in spectrophotometer.
15. The step 13 was repeated as 5 times to get the average value for the protein absorbance.
16. The protein concentration was calculate using formula y=0.1276 ln(x) + 0.0346 for Biuret
method and y= 0.0405x - 0.015 for Lowry method.
DISCUSSION ON IT
The protein concentration in freshwater fish is more than the marine fish. The marine water live in salty conditions differ from habitat of freshwater fish. So, the marine fish has to balancing the body fluid by regulate the osmoregulation. The fish has high tendency to release water out of the body by osmotic regulation. Together by that the fish also excrete more salt through gill such as ammonia. Ammonia is the ammonium group in amino acid. Thats why the protein concentration in marine fish is less than the freshwater fish.
The method use between Biuret and Lowry show the Lowry method is more accurate to measure the protein concentration in fish. This is because when comparing our result with the others, the marine fish (Kayu Hitam) has too much difference in protein concentration result since this sample is tested by using Biuret method. My result is x=17.3993A and one of the other group result is x=49.6976A. For Lowry method, the reading of absorbance is more accurate since Lowry method is more sensitive since it combines the reaction of copper ion with the peptide bonds under alkaline conditions. Freshwater fish (Talapia merah) protein concentration of mine is x=24.4444A while the other group result is x=28.8519A.
The method use between Biuret and Lowry show the Lowry method is more accurate to measure the protein concentration in fish. This is because when comparing our result with the others, the marine fish (Kayu Hitam) has too much difference in protein concentration result since this sample is tested by using Biuret method. My result is x=17.3993A and one of the other group result is x=49.6976A. For Lowry method, the reading of absorbance is more accurate since Lowry method is more sensitive since it combines the reaction of copper ion with the peptide bonds under alkaline conditions. Freshwater fish (Talapia merah) protein concentration of mine is x=24.4444A while the other group result is x=28.8519A.
NEW THINGS THAT I LEARNED
1. I learned how to handle spectrophotometer.
2. I know to use micropippete.
3. I can applied the formula of Biuret and Lowry method to calculate the protein concentration.
2. I know to use micropippete.
3. I can applied the formula of Biuret and Lowry method to calculate the protein concentration.
THE BEST THINGS I WANT TO KNOW
The best thing I want to know is about the the protein concentration of fish that have 2 habitat means sometimes they live in the ocean and sometimes in freshwater such as stickleback fish(Gasterosteus aculeatus).