Name : Clodagh Coughlan
Module: Structural Biochemistry
Date of experiment :
Date of submission: 14/11/18
Experiment title: Biuret assay to determine protein concentration
Lecturer: Lisa MeaneyAims and objectives:
The of this experiment was the use of Biuret assay to determine the concentration of protein in BSA and determining the absorption by using a spectrophotometer.
There are three assays that can be used to determine the concentration of protein in a sample. They are biuret assays , Bradford’s assays and Lowry’s assays. The biuret assays was used in this experiment. It is a test that is used for a protein that has more than 2 peptide bonds in a sample. The blue biuret agent added will turn violet if there is more than 2 peptide bonds in the sample. The protein forms a complex with Cu2+ in an alkaline solution(Clinical Biochemistry of domestic animals1). The solution is analysed at 540nm on the spectrophotometer after the violet colour was obtained. The advantage to this method is that very little materials interfere with it like amino acid buffers. Also, results can be obtained quickly and they are reliable and accurate. It is also cost effective and non-toxic.
Bradford’s assays is a very easy to use and provides accurate results for determining the concentration of protein in a solution. However it is ineffective when using BSA and bovine gamma-globulin as the reagents show little or no interference with the protein. (Bio-Rad laboratories Inc3)
Lowry assays is another method for determining the level of protein in a given solution. Copper is used in alkaline conditions, this forms monovalent copper ins that then react with the Folin reagent. The protein concentration is observed by a blue colour changes which can then be measured using a spectrophotometer.
Bovine Serum Albumin (BSA) is a serum albumin protein that is derived from cows. It is commonly used in lab experiments because of its stability to increase signal in assays, it is cheap as it is readily available. (Rockland antibodies and assays4)
The absorbance of the standard solutions and the samples were measured using the spectrophotometer and had a set wavelength of 540nm.
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Materials and methods:
As per lab manual pages 22-23.
Protein sample used BSA.
Table 1: Sample preparation for determining the protein concentration of BSA.
Test tube no. Ml % BSA Mls of water Mg BSA in 1 ml Absorbance:
1 0 1.0 0 0.075
2 0.25 0.75 2.5 0.166
3 0.50 0.50 5.0 0.249
4 0.75 0.25 7.5 0.355
5 1.0 0 10 0.442
6 0.5 0.5 7.92 0.290
7 0.3 0.7 5.960.218
Figure 1: BSA standard curve.
Calculations for unknown concentration:
equation of the line:
concentration for test tube 6 is 7.92
Test tube 7:
Concentration of test tube 7 is 5.96
From analysing the results and the graph it’s clear that the concentration increases proportionally to the absorbance. The unknown concentrations were found by using the equation if the line. The absorption values of the unknown solutions were test tube 6 had a concentration of 7.92 and test tube 7 had a concentration of 5.96. Furthermore, this also proves that as the concentration of the BSA increases the absorbance increase proportionally.
The chemical group which gives a colour with Biuret regent is the peptide bonds. A peptide bond is formed between the carboxyl group of one molecule and the amino group of another molecule that releases a water molecule in the process.
The limitations for this assays were that it has a low sensitivity, the sample being tested must contain at least 1 mg of protein for this assays to be effective. Also a limitation with the biuret assays is that compounds like Tris buffers and ammonium sulphate can interfere with the development of the violet colour. ( Onuedu.2018)
1 Book: P. David Eckersall,’Clinical biochemistry of domestic animals’ sixth edition (2008)
2 James E. Noble, Marc J.A Bailey, ‘Methods in enzymology'(2009) can be found at: https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/biuret-test
3 (Online) Dr. Alfred Nobel ‘Bio-Rad Laboratories Inc.
Can be found at: http://www.bio-rad.com/webroot/web/pdf/lsr/literature/4110065A.pdf
4 (Online): Rockland antibodies and assays ‘Bovine serum albumin’
Can be found at: https://rockland-inc.com/bovine-serum-albumin.aspx
5 Onuedu. 2018. Onuedu. Online. Available from: http://www2.onu.edu/~k-broekemeier/Chem314/ReviewProteinMethods.pdf