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Developing purification protocols
The development of a purification protocol involves four basic phases:
1. Forming a platform for the development
The platform should include:
- Intended quantity and quality of the end product.
- Available information on the physical properties of the target protein.
- Available information on the type of starting material and content of the target protein.
- Information on the range of conditions compatible with sample stability (the sample stability window, SSW).
2. Running initial purification experiments.
The Standard Purification Protocol is used as a starting point for the development. In many cases, this will provide sufficient purity, while in others it may need to be further optimized.
3. Optimizing the purification protocol.
Guided by the outcome of the standard protocol experiment, the purification steps are optimized and/or recombined until satisfactory results are obtained.
4. Adapting the scale of the final purification protocol.
Normally it is practical to run initial and optimization experiments on small columns in HiTrap™ format. If enough purified material cannot be obtained with HiTrap columns , the protocol is scaled up according to certain simple rules.
Fig 2.1. Workflow of purification protocol development. |
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