Designing a Separation

The goal of a protein separation is to obtain the protein in a pure, active form in the minimum number of steps and the shortest time possible. Let's examine some significant words in that first statement.

Pure: To properly characterize a protein, it must be completely isolated from all other proteins and cellular components. There are many proteins in living cells, so any individual protein is not likely to comprise more than 1% of the total protein in a cell. To purify a protein, then you must remove about 99% of the total protein that is present in your original source material.

Active: Proteins are designed to function in living, dynamic systems. The act of purifying a protein involves removing it from its native environment. Therefore, when proteins are purified attempts are usually made to maintain certain conditions that may be present in its normal environment, such as pH, salt concentration, and reducing conditions.

Minimum number of steps: Each step in a protein purification procedure typically reduces the overall yield of the pure protein you will obtain at the end of your separation scheme. Additionally, each extra step requires more time and may involve additional costs for equipment and supplies.

Shortest time: In living systems, proteins are designed to function for a short period of time and then be recycled by the cell. This built in lack of stability leads to the loss of activity in many proteins in the purification process. The more quickly the protein can be purified and stored under stable conditions, the better chance that you will be able to characterize and active protein. Final storage conditions often involve flushing the samples with an inert gas to prevent air oxidation of the protein, storage at -70 ° C, and the presence of additives such as EDTA (to trap metal ions that might damage a protein), and sugars such as glycerol which often lend some stability to proteins.

Several factors need to be considered when designing a protein purification

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