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Prepare the columnPerform a blank run (use binding buffer instead of sample) to ensure that loosely bound ligand isremoved (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.Purification1.Apply sample. Optimal flow rate is dependent on the binding constant of the ligand, but a recommendedflow 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 isweak, 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 prepackeddesalting columns (see Chapter 9).Reequilibrate the columnReequilibrate the column by washing with 10 column volumes of binding buffer.
174 Handbook 18-1142-75AC
Handbook 18-1142-75AC 175StepPurityCaptureIntermediatepurificationPolishingPreparation,extraction,clarificationAchieve finalhigh level purityRemove bulkimpuritiesIsolate, concentrateand stabilizeChapter 7Multistep purification of tagged and untaggedrecombinant proteinsRecombinant protein expression may allow production of large amounts of an affinity-tagged proteinso that a single purification step using affinity chromatography is sufficient to achieve the desired levelof purity. However, the purification obtained after a single step is frequently not sufficient, and affinitytags may sometimes interfere with the post-purification use of the protein. In these instances, multisteppurification will be necessary.A significant advantage when working with recombinant proteins is that there is often considerableinformation available about the product (amino acid sequence, Mr, pI, functional properties) andcontaminants (the expression host may be well known). With this information, detection assays andsample preparation and extraction procedures in place, a purification strategy of Capture, IntermediatePurification, and Polishing (CIPP) can be applied (Figure 74). This strategy is used in both the pharmaceuticalindustry and in the research laboratory to ensure faster method development, a shorter time to pureproduct, 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 discussedherein. The Protein Purification Handbook(from GE Healthcare) is recommended as a guide to planningefficient and effective protein purification strategies.