Estimation of protein concentration
Experiment 2: Estimation of protein concentration
Introduction
Protein assays are designed to measure the total protein in a solution. Protein assays are quantitative if the protein to be assayed is available in sufficient quantity such that one is able to use it to create a standard curve. If this cannot be achieved, then a standard protein, such as albumin, may be used for a standard curve with the understanding that the results on the unknown protein are semi-quantitative. Since most proteins are not available in large quantities, standard curves for protein assays are typically based on the use of either bovine serum albumin (BSA) or bovine gamma globulin (IgG). The Bradford protein assay is a method to determine protein concentration in the mixture. This method is based on the proportional binding of the dye Coomassie to proteins. If there is a higher protein content, the more Coomassie binds and it produces a significant change in colour of the mixture. The Bradford protein assay contains the dye Coomassie Blue G-250 which is red brown colour in acidic solution. When the protein binds to the dye, the pKa of the dye shifts and causes the dye to turn blue. The maximum absorbance spectrum of the dye is at 595nm. According to Beer-Lambert Law, a standard curve of absorbance versus protein concentration produced by measuring the absorbance of the protein solutions of a known concentration is often used to estimate the protein concentration of an unknown solution. Other than that, the Bradford protein assay reacts variedly with the composition of the protein and is also sensitive to non-protein sources, particularly detergents, and becomes nonlinear with higher protein concentrations. Objectives
1. To prepare a standard curve of absorbance versus protein concentration by using Bovine Serum Albumin (BSA)
2. To determine BSA concentration in two sample solutions
3. To determine protein concentration in apple juice.
Materials
Bradford reagent,
Introduction
Protein assays are designed to measure the total protein in a solution. Protein assays are quantitative if the protein to be assayed is available in sufficient quantity such that one is able to use it to create a standard curve. If this cannot be achieved, then a standard protein, such as albumin, may be used for a standard curve with the understanding that the results on the unknown protein are semi-quantitative. Since most proteins are not available in large quantities, standard curves for protein assays are typically based on the use of either bovine serum albumin (BSA) or bovine gamma globulin (IgG). The Bradford protein assay is a method to determine protein concentration in the mixture. This method is based on the proportional binding of the dye Coomassie to proteins. If there is a higher protein content, the more Coomassie binds and it produces a significant change in colour of the mixture. The Bradford protein assay contains the dye Coomassie Blue G-250 which is red brown colour in acidic solution. When the protein binds to the dye, the pKa of the dye shifts and causes the dye to turn blue. The maximum absorbance spectrum of the dye is at 595nm. According to Beer-Lambert Law, a standard curve of absorbance versus protein concentration produced by measuring the absorbance of the protein solutions of a known concentration is often used to estimate the protein concentration of an unknown solution. Other than that, the Bradford protein assay reacts variedly with the composition of the protein and is also sensitive to non-protein sources, particularly detergents, and becomes nonlinear with higher protein concentrations. Objectives
1. To prepare a standard curve of absorbance versus protein concentration by using Bovine Serum Albumin (BSA)
2. To determine BSA concentration in two sample solutions
3. To determine protein concentration in apple juice.
Materials
Bradford reagent,
References: Protein Assay using Bradford method http://www.scribd.com/doc/51367139/Protein-Assay-Using-Bradford-Method Selecting a protein assay http://www.piercenet.com/guide/protein-assay-selection-guide The Bradford Assay http://liferaydemo.unl.edu/web/biochem/BradfordAssaySite