Prepare the column Perform a blank run use binding buffer instead of sample to

Prepare the column perform a blank run use binding

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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.
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174 Handbook 18-1142-75AC
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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.
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