experiment 1: PROTEIN ASSAY
Experiment 1: Protein assay
Obejctive:
Method:
Result:
Obejctive:
- To compare the freshwater and marine fishes's protein concentration
- To determine and compare the concentration of Kayu Hitam fish and Talapia fish
- To study the reasons on why these fishes have different concentration of protein.
Method:
- First of all, we prepared the solution of gelatines at 1, 2, 3, 4, 5 and 6 mg/mL in water of Biuret assay to get standard graph of protein concentration.
- We calculated the volume of gelatines needed for dilution to form different concentration of gelatines as stated before. The formula used is M1V1=M2V2. For example, to calculate the volume of 5mg/mL gelatines, we substituted M1=6mg/mL, M2=5mg/mL, and V2=10mL to determine V1. So, here V1 is equal to 8.33mg/mL of gelatines.
- Then, we added 1.7mL of distilled water for dilution to form 5mg/mL gelatines. This calculation is applied to prepare the rest different concentration of gelatines.
- Plus, we used pipette, 10mL measuring cylinder, beakers and dropper to prepare the solutions.
- After that, we measured 0.5mL of all the proteins with different concentration and added Biuret reagent each. The colour changed from colourless into dark blue.
- We left them for 10 minutes before we measure the absorbance of each protein.
- To calculate absorbance of the standards and the freshwater and marine fish’s samples, we used spectrophotometry. 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.
- This is my first time using the spectrophotometer and I was quite excited about it.
- Then, 1mL of each gelatine with different concentration is put into the spectrophotometry 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 same steps is 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.
- Now, we have two types of graph.
- Next, for Biuret method, the sample (Kayu Hitam fish and Talapia fish) 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.
Result:
So, from the table/ graph shows that gelatine that contain higher concentration will have higher absorbance but because Lowry assay method rely on the presence of readily oxidisable amino acids such as tyrosine, cysteine & tryptophan there is a variation in response from proteins with differing amino acid content. An overabundance of the amino acids in relation to the assay reagents as would occur with high protein level, will result in a loss of linearity of the assay.
In this experiment, we have to investigate about the protein contents (concentration) in the two fishes's samples which are ikan talapia and ikan kayu hitam. We used Biuret reagent test to test the content of protein in all those fishes samples. The Biuret test is a chemical test used for detecting the presence of peptide bonds. In the presence of peptides, a copper(II) ion forms violet-colour coordination complexes in an alkaline solution. The Biuret reaction can be used to assess the concentration of proteins because peptide bonds occur with the same frequency per amino acid in the peptide. The intensity of the colour and hence the absorption at 540 nm is directly proportional to the protein concentration according to the Beer-Lambert law.
We found that the concentration of protein in the Talapia fish (freshwater fish) is pretty higher compared to Kayu Hitam fish (marine fish). Based on Biuret method for Kayu Hitam fish, its absorbance is 0.399A while in Lowry method for Talapia fish, its absorbance is 0.975A. Based on Biuret equation which is y=0.1276ln(x)+0.0346 and the protein concentration in Kayu HItam fish is 17.3993A. While Lowry equation is y=0.0405x-0.015 and the protein concentration of Talapia fish is 24.444A.
In this experiment, we have to investigate about the protein contents (concentration) in the two fishes's samples which are ikan talapia and ikan kayu hitam. We used Biuret reagent test to test the content of protein in all those fishes samples. The Biuret test is a chemical test used for detecting the presence of peptide bonds. In the presence of peptides, a copper(II) ion forms violet-colour coordination complexes in an alkaline solution. The Biuret reaction can be used to assess the concentration of proteins because peptide bonds occur with the same frequency per amino acid in the peptide. The intensity of the colour and hence the absorption at 540 nm is directly proportional to the protein concentration according to the Beer-Lambert law.
We found that the concentration of protein in the Talapia fish (freshwater fish) is pretty higher compared to Kayu Hitam fish (marine fish). Based on Biuret method for Kayu Hitam fish, its absorbance is 0.399A while in Lowry method for Talapia fish, its absorbance is 0.975A. Based on Biuret equation which is y=0.1276ln(x)+0.0346 and the protein concentration in Kayu HItam fish is 17.3993A. While Lowry equation is y=0.0405x-0.015 and the protein concentration of Talapia fish is 24.444A.