Protein Purification Made Easy

Friday, 01 January, 2021

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Proteins can be isolated based on differences in their physical and chemical properties, and can be analysed and purified using techniques like centrifugation, electrophoresis and chromatography — all of which have different benefits and outcomes.

Tagged recombinant proteins are relatively straightforward to purify. We recommend researchers use an affinity resin that corresponds to the tag system used and the natural conditions for the protein to avoid precipitation and degradation. Two-step purification is commonly used, the first step being the use of an affinity chromatography resin and the second step using a size-exclusion chromatography resin. If your requirement for purity is high, add an additional intermediate step of ion exchange or hydrophobic interaction chromatography to increase purity. However, try to use as few steps as possible as adding steps will decrease overall protein yield.

After purification, you may need to cleave off the tag before using the protein. The tag-free target protein needs to be separated from the tag and target protein still containing the tag. On-column cleavage can also be used where protease is added to the column with the target protein bound, enabling cleavage and removal to be performed in one step.

For non-tagged proteins, more effort is required to develop the protocols. Experimental planning is important to make conscious decisions for what conditions to test, what to optimise, and the requirements for purity and yield. In our experience, successful protein purification requires following a multi-step approach to perform Capture, Intermediate Purification and Polishing (CiPP).

To control your protein purification and deliver significantly more information on your target protein and impurities we recommend the use of a protein purification system from our ÄKTA system range. If you are new to protein purification or looking for tips on how to run and take care of your ÄKTA system, our Cytiva team has put together a set of free online training modules. Learn more at

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