To Determine the Concentration of Protein in a Solution Using Biuret Method
Essay by Ðhruv Kaushik • April 21, 2016 • Lab Report • 534 Words (3 Pages) • 1,122 Views
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To determine the concentration of protein in a solution using Biuret method.
Introduction
One commonly used method for determining the total protein in a sample is the Biuret method. The Biuret method is based on the complexation of Cu2+ to functional groups in the protein’s peptide bonds as shown in Figure 1. The formation of a Cu2+-protein complex requires two peptide bonds and produces a violet-colored chelate product which is measured by absorption spectroscopy at 540 nm (Newton & Hollinger, 1979). Over a given concentration range, the measured absorption at 540 nm is linear with respect to the concentration of total protein. This relationship allows a standard curve to be created that is used to calculate the concentration of an unknown sample (Newton & Hollinger, 1979).
[pic 1]
Figure No 1: Biuret reagent reacts with an alkaline solution of CuSO4 to form a violet chelate compound.
Method
6 BSA standard solution has been prepared in the range 0-10 mg/mL and labelled, The tube containing no protein should contain 1 mL of Distilled Water.
4 mL of biuret reagent is added to each standard solution and unknown sample solution and kept for 30min to stand and absorbance has been recorded at 540nm.
Calibration graph has been plotted, the absorbance against protein concentration on X axis and using calibration curve equation concentration of protein in unknown solution has been calculated.
Results and Discussion
Table No 1: Table showing content and absorbance of each standard protein solution
Protein Concentration (mg/mL) | 0 | 2 | 4 | 6 | 8 | 10 |
BSA Solution (mL) | 0 | 0.2 | 0.4 | 0.6 | 0.8 | 1 |
Water (mL) | 1 | 0.8 | 0.6 | 0.4 | 0.2 | 0 |
Biuret reagent (mL) | 4 | 4 | 4 | 4 | 4 | 4 |
Absorbance at 540nm | 0 | 0.173 | 0.28 | 0.374 | o.430 | 0.517 |
Table no 2: Table showing Absorbance of unknown solution and calculated concentration.
Unknown Solution | 1 | 3 |
Absorbance at 540nm | 0.212 | 0.395 |
Protein Concentration (mg/mL) | 3.30 | 7.01 |
[pic 2]
Graph No.1: Graph absorbance against the known protein Concentration on X axis.
Concentration of unknown sample no. 1
Y=0.0493x + 0.0492
0.212=0.0493x + 0.0492
X= 3.30 mg/mL
Concentration of unknown sample no. 1
Y=0.0493x + 0.0492
X = 7.01 mg/mL
The subunits which make up proteins are amino acids. The amino acids are joined together by dehydration synthesis to forms chains, which are hundreds of amino acids long which is called proteins. Proteins function as enzymes or as structural units in cells. They do most of the work in a cell. Almost all of the exciting stuff such as metabolism, memory, hormone action, and movement involves proteins. In this lab, we have learnt method of measuring protein concentration, biuret assay.
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