Tip of the Week

#6 What Factors Should You Consider for Chromatography Method Development

Know relevant properties of your protein: pI, molecular weight, hydrophobicity. This will assist in developing a purification strategy.

If using IMAC as a capture step, do not add EDTA to your buffers. These will strip the active metals and preclude protein binding.

When creating methods try to minimize redundant steps for clarity of protocol and ease of monitoring. Creating method templates saves future setup time and eliminates the need to monitor entire runs.

Tris-based buffers should not be used in IMAC purification, as they will outcompete the protein of interest for the binding sites.

Do not use phosphate buffer systems when using hydroxyapatite resins, as they will interfere with protein binding

Always name your runs by date or notebook number to facilitate easy data access in the future.

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