Isolation: Proteins are large molecules with diverse biological functions. They could be catalysts, receptors, chemical messengers etc. in order to study proteins, they need to be isolated in pure form. The three-dimensional structure of most proteins is a consequence of many relatively weak non-covalent interactions. Disrupting cellular structure is required to release the proteins from the cell. Purification of proteins begins with homogenizing the tissues, then subsequent fractionation and purification of cellular constituents. In this experiment, the source of protein was sprouting seeds. They were homogenized using a sodium phosphate buffer, pH 7, in a blender. After filtration, it was centrifuged. The supernatant was then isolated and used for fractionation.
Fractionation: Precipitation of proteins from a solution may be accomplished by fractional precipitation techniques using salts, organic solvents or pH. The first method of protein precipitation was by using salts. Sodium phosphate with pH 7 was used. Most common salts are ammonium sulfate and sodium sulfate. At low concentration of the salt, the solubility of the proteins usually increases because of salting in effect. When the concentration of salt is high, protein solubility will decrease then the protein will be salted out. When a large amount of salt
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Determining an effective assay is often difficult; but the more specific the assay, the more effective the purification. For enzymes, which are protein catalysts the assay is usually based on the reaction that the enzyme catalyzes in the cell. In this experiment, amylase enzyme is tested. it hydrolyzes starch to monosaccharides. The amylose component of starch complexes with iodine as follows and produces blue to purple complex. Hydrolysis of starch by amylase enzyme would result in the disappearance of the