Protein purification is an activity that has occupied the time of biochemists throughout the history of biochemistry

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Protein purification is an activity that has occupied the time of biochemists throughout the history of biochemistry. In fact, a large percentage of the biochemical literature is a description of how specific proteins have been separated from the thousands of other proteins and other biomolecules in tissues, cells and biological fluids. Biochemical investigations of all biological processes require, at some time, the isolation, purification and characterization of a protein. Of course, there is no single technique or sequence of techniques that can be followed to purify all proteins. Most investigators approach the problem by trial and error. Fortunately, the experiences and discoveries of many biochemists have been combined so that we have a general and somewhat systematic approach to the purification of proteins. The best purification procedure is one that yields a maximum amount of the purified protein in a minimum amount of time.

?-lactalbumin, a protein found in milk is important for the synthesis of lactose. It is one of the components of the lactose synthase system. The protein can be isolated readily by a variety of techniques including salt precipitation, gel filtration or affinity chromatography. This experiment introduces the student to a series of protein purification steps that yield ?-lactalbumin in sufficient amounts and purity for further characterization by UV spectroscopy and methods like SDS-polyacrylamide gel electrophoresis.

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? -lactalbumin has a molecular weight of 15,500 and contains 129 amino acid residues. Normally it is present in a concentration of 1mg/ml in fresh milk. The main proteins found in fresh milk are the caseins which can be removed by acid and heat-induced precipitation at pH 4.6, or the addition of sodium sulphate. ?-lactalbumin can be precipitated out by lowering the pH to 4.0. In this experiment, you will attempt to isolate and purify ?-lactalbumin from fresh milk using the technique of acid precipitation.


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