Preliminary considerations

To achieve successful results, the total separation problem must be considered from the beginning and. as more information accrues, the procedures in use must be frequently reassessed. The first step is to obtain as much information as possible from the current literature on the protein of interest and, secondly, to define the purpose of the purification. For example: Are large amounts required? What degree of purity is needed? It is then possible to set minimum objectives for the end-product in terms of purity, yield and economy. Decisions can now be made regarding more practical details:

1. Selection of source: primary considerations when selecting source material include ease of extraction together with quantity and stability of the protein within the material itself.

2. Assay development: throughout the separation, it is essential to determine total protein in the sample using routine assays such as Lowry or Bradford together with assaying for activity of the specific protein. These assays provide constant monitoring of recovery and reveal immediately any sudden loss of activity. This information can be used to modify and improve procedures.

3. Protein stability: both investigation of source material and development of an activity assay will yield information on the stability of the target protein. It is vital to be aware at an early stage of any protein sensitivity to temperature, pH, organic solvents, ionic strength, metal ions, inhibitors or contact surfaces; any one of these may result in significant loss of activity and should therefore be taken into account during development work. Avoidance of protein disruption during initial extraction procedures will be discussed below.

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