Affinity Chromatography - Department of Molecular and Cellular ...

Appendix 3
Column packing and preparation
Prepacked columns from Amersham Pharmacia Biotech will ensure reproducible results and
the highest performance. However, if column packing is required, the following guidelines
will apply at any scale of operation:
With a high binding capacity medium, short, wide columns can be used for rapid purification,
even with low linear flow rates.
Ready to use affinity media are supplied with details of the binding capacity per ml of
medium. Unless otherwise stated, estimate the amount of medium required to bind the
target molecules and use two- to five times this amount to pack the column. Refer to the
product instructions for more specific information regarding buffers, flow rates etc.
For affinity media made from pre-activated matrices, determine the binding capacity of the
medium. Estimate the amount of medium required to bind the target molecules and use
two- to five times this amount to pack the column.
A Column Packing Video is available to demonstrate how to produce a well-packed column
(see Ordering information). The video focuses particularly on the importance of column
packing for gel filtration, shown in the figure below.
1. Equilibrate all materials to the temperature at which the purification will be performed.
2. Eliminate air by flushing column end pieces with the recommended buffer. Ensure no air is trapped under the
column net. Close column outlet leaving 1–2 cm of buffer in the column.
3. Gently resuspend the purification medium.
For media not supplied in suspension, use a medium: buffer ratio of approximately 1:2 to
produce a suspension for mixing during rehydration.
Avoid using magnetic stirrers since they may damage the matrix.
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