Prepare the column perform a blank run use binding

Prepare the column
Perform a blank run (use binding buffer instead of sample) to ensure that loosely bound ligand is
removed (see below).
1. Wash with 3 column volumes of binding buffer.
2. Wash with 3 column volumes of elution buffer.
3. Equilibrate with 5 to 10 column volumes of binding buffer.
Purification
1. Apply sample. Optimal flow rate is dependent on the binding constant of the ligand, but a recommended
flow rate range is, for example, 0.2 to 1 ml/min on a HiTrap 1-ml column.
2. Wash with 5 to 10 column volumes of binding buffer, or until no material appears in the eluent.
Avoid excessive washing if the interaction between the protein of interest and the ligand is
weak, because this may decrease the yield.
3. Elute with 2 to 5 column volumes of elution buffer.
4. If required, purified fractions can be desalted and exchanged into the buffer of choice using prepacked
desalting columns (see Chapter 9).
Reequilibrate the column
Reequilibrate the column by washing with 10 column volumes of binding buffer.

174 Handbook 18-1142-75AC

Handbook 18-1142-75AC 175
Step
Purity
Capture
Intermediate
purification
Polishing
Preparation,
extraction,
clarification
Achieve final
high level purity
Remove bulk
impurities
Isolate, concentrate
and stabilize
Chapter 7
Multistep purification of tagged and untagged
recombinant proteins
Recombinant protein expression may allow production of large amounts of an affinity-tagged protein
so that a single purification step using affinity chromatography is sufficient to achieve the desired level
of purity. However, the purification obtained after a single step is frequently not sufficient, and affinity
tags may sometimes interfere with the post-purification use of the protein. In these instances, multistep
purification will be necessary.
A significant advantage when working with recombinant proteins is that there is often considerable
information available about the product (amino acid sequence, M
r
, pI, functional properties) and
contaminants (the expression host may be well known). With this information, detection assays and
sample preparation and extraction procedures in place, a purification strategy of Capture, Intermediate
Purification, and Polishing (CIPP) can be applied (Figure 74). This strategy is used in both the pharmaceutical
industry and in the research laboratory to ensure faster method development, a shorter time to pure
product, and good economy.
This section gives a brief overview of the approach recommended for any multistep protein purification.
Appendix 9 provides useful background information describing the various techniques discussed
herein. The Protein Purification Handbook (from GE Healthcare) is recommended as a guide to planning
efficient and effective protein purification strategies.