To assure the maximum yield and to avoid unnecessary denaturation of the enzymes, most of the protein purification work is usually carried out at low temperatures, i.e. between 0 and 40ºC. However, it is simply far more convenient to work in a regular laboratory room as opposed to a cold room. Since the purpose of this experiment is to demonstrate the use of common purification techniques, unless noted otherwise when it is truly critical, the procedures will be carried out at room temperature without any significant loss of educational values.
The recovery of protein can have very significant economical implications. Because a fixed fraction of the original protein stays soluble in the solution, the recovery of protein is often not near 100%. Of course, a yield of over 100% indicates that there may be problems associated with the assay method.
In a typical protein preparation or purification step carried out in a laboratory where the aim is to isolate a small quantity of a product for structural or kinetic studies, a saturated ammonium sulfate solution is routinely used. It is also the procedure taken in this experiment. However, in an actual large scale commercial process, it is better to add ammonium sulfate directly into the protein mixture as powdered solids so that the effect of dilution by the salt solution is minimized.
To assure the maximum yield and to avoid unnecessary denaturation of the enzymes, most of the protein purification work is usually carried out at low temperatures, i.e. between 0 and 40ºC. However, it is simply far more convenient to work in a regular laboratory room as opposed to a cold room. Since the purpose of this experiment is to demonstrate the use of common purification techniques, unless noted otherwise when it is truly critical, the procedures will be carried out at room temperature without any significant loss of educational values.The recovery of protein can have very significant economical implications. Because a fixed fraction of the original protein stays soluble in the solution, the recovery of protein is often not near 100%. Of course, a yield of over 100% indicates that there may be problems associated with the assay method.In a typical protein preparation or purification step carried out in a laboratory where the aim is to isolate a small quantity of a product for structural or kinetic studies, a saturated ammonium sulfate solution is routinely used. It is also the procedure taken in this experiment. However, in an actual large scale commercial process, it is better to add ammonium sulfate directly into the protein mixture as powdered solids so that the effect of dilution by the salt solution is minimized.
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