Protein Assay
OBJECTIVES:
To measure protein concentration by the biuret method and Lowry method using fishes.
INTRODUCTION:
The determination of protein concentration is an essential technique in all aspects of protein studies.This lab activity is designed to teach students the principles behind a common protein estimation assay known as the Biuret Protein Assay and Lowry Protein Assay.
To measure protein concentration by the biuret method and Lowry method using fishes.
INTRODUCTION:
The determination of protein concentration is an essential technique in all aspects of protein studies.This lab activity is designed to teach students the principles behind a common protein estimation assay known as the Biuret Protein Assay and Lowry Protein Assay.
BIURET METHOD
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 color, and hence the absorption is directly proportional to the protein concentration, according to the Beer-Lambert law. |
LOWRY METHOD
The Lowry protein method can be used to detect amount of protein. Its more sensitive, highly reproducible, inexpensive and easy to perform than biuret method. |
PROCEDURE
sample preparation 1
-we macerate fish in smaller size and blend inphosphate buffer saline.
-incubate 24hours at 4c in shaker
sample preparation 2
-sample protein we filter it and centrifuge 15minute in shaker
-Biuret assay we prepared gelatin at 1 until 6mg/mL in water
-Lowry assay we prepared gelatin 0.1 until 0.6mg/L
-Biuret method, 0.5mLprotein added 2.5mL reagent and incubate 10minute then read at 540nm
-Measured absorbance and plot standard curve
-Lowry method,0.25mL protein added 2.5mL Lowry reagent and incubate 10minute.After that we added 0.25mL Lowry reagent 2 then incubate 30minute then read at 750nm.
-Measure the absorbance and compare the concentration using both methods.
MATERIALS
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 ofEuthynnus 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