Exp 4
Title: Isolation and purification of bovine milk α-lactalbumin.
Abstract:
The Bradford assay, a colorimetric protein assay, is based on an absorbance shift of the dye Coomassie Brilliant Blue G-250 in which under acidic conditions the red form of the dye is converted into its bluer form to bind to the protein being assayed. The (bound) form of the dye has an absorption spectrum maximum historically held to be at 595 nm.
Objective:
To determine the technique to a series of protein purification steps that yield α-lactalbumin in sufficient amounts and purity for futher characterization by UV spectroscopy.
Introduction:
Protein purification is a series of processes intended to isolate a single type of protein from a complex mixture. Protein purification is vital for the characterization of the function, structure and interactions of the protein of interest. The various steps in the purification process may free the protein from a matrix that confines it, separate the protein and non-protein parts of the mixture, and finally separate the desired protein from all other proteins. Separation of one protein from all others is typically the most laborious aspect of protein purification. The most abundant protein found in milk is caseins and it can be removed by heat and low pH. The most general method to monitor the purification process is by running a SDS-polyacrylamide gel electrophoresis of the different steps. This method only gives a rough measure of the amounts of different proteins in the mixture, and it is not able to distinguish between proteins with similar apparent molecular weight. the purest, isolated form of bovine alpha-lactalbumin commercially available. Alpha-lactalbumin is the primary protein in human milk, and is therefore extremely important for infant nutrition. The structure of alpha-lactalbumin is well known and is composed of 123 amino acids and 4 disulfide bridges. The molecular weight of alpha-lactalbumin is 14.2K Daltons. Alpha
Abstract:
The Bradford assay, a colorimetric protein assay, is based on an absorbance shift of the dye Coomassie Brilliant Blue G-250 in which under acidic conditions the red form of the dye is converted into its bluer form to bind to the protein being assayed. The (bound) form of the dye has an absorption spectrum maximum historically held to be at 595 nm.
Objective:
To determine the technique to a series of protein purification steps that yield α-lactalbumin in sufficient amounts and purity for futher characterization by UV spectroscopy.
Introduction:
Protein purification is a series of processes intended to isolate a single type of protein from a complex mixture. Protein purification is vital for the characterization of the function, structure and interactions of the protein of interest. The various steps in the purification process may free the protein from a matrix that confines it, separate the protein and non-protein parts of the mixture, and finally separate the desired protein from all other proteins. Separation of one protein from all others is typically the most laborious aspect of protein purification. The most abundant protein found in milk is caseins and it can be removed by heat and low pH. The most general method to monitor the purification process is by running a SDS-polyacrylamide gel electrophoresis of the different steps. This method only gives a rough measure of the amounts of different proteins in the mixture, and it is not able to distinguish between proteins with similar apparent molecular weight. the purest, isolated form of bovine alpha-lactalbumin commercially available. Alpha-lactalbumin is the primary protein in human milk, and is therefore extremely important for infant nutrition. The structure of alpha-lactalbumin is well known and is composed of 123 amino acids and 4 disulfide bridges. The molecular weight of alpha-lactalbumin is 14.2K Daltons. Alpha