Protein Purification(PP 1)

There are various methods that separate proteins according to distinct chemical and physical properties. Proteins can be purified by sequentially applying these techniques. Before purifying a protein one needs to consider how much protein is needed, how pure it needs to be, whether the purified protein needsto be in its native configuration, or express an activity.

These considerations will be determined by the ultimate use for the purified protein. If the purified protein is going to be used in functional and structural studies,isolating the protein in its native and active configuration will be important. However, if the goal is to determine a partial amino acid sequence then concerns about denaturation are minimal.

The optimal method to purify a protein is empirically determined by carrying out smallscale pilot studies. After the optimal conditions for a particular purification step is worked out, then the procedures can be scaled up. The order in which the steps are carried out will also affect the final outcome. In general, though, it is best to start with high capacity methods that are easily and quickly carried out and then proceed to high resolution/low capacity methods.

Capacity refers to how much total protein can be readily accommodated and
resolution refers to the ability to separate the protein of interest from contaminating proteins. Generally, as the resolution of the technique increases,
the time required to carry out the technique increases and the protein capacity decreases. One exception is that gel filtration is not usually ahigh resolution technique, as well as being low capacity and relatively difficult to carry out.

The capacity and resolution of affinity chromatography depends on the ligand. Some ligands afford both high capacity and high resolution. The resolution of any of the chromatographic technique can be substantially enhanced through the use of HPLC. However, the capacity of HPLC columns is much lower so that it may not be useful until later in the purification protocol.