Purification methods are selected from affinity chromatography, ion exchange, size exclusion, hydrophobic interaction and reversed-phase techniques.
Purity targets and buffer conditions are defined with the research application in mind. An ELISA antigen and a crystallography sample are not purified the same way.
A typical tagged protein uses affinity capture, a polishing ion-exchange or SEC step, and buffer exchange into the customer-specified storage buffer. We document the steps that were actually run.
Purity is not a single number copied from peptide HPLC. SDS-PAGE, a chromatogram and, when requested, mass confirmation describe different things. State which readout the assay needs.
Storage buffer, glycerol, reducing agent and freeze–thaw instructions belong in the same conversation as the purification train. A protein that looks clean on a gel can still fail if it is frozen in the wrong buffer.
Choosing the train
His-tagged intracellular proteins often start with immobilized-metal affinity chromatography. Secreted Fc fusions may use Protein A. Untagged proteins need a method that the construct actually supports. We will not invent a universal three-column recipe.
What polishing is for
Ion exchange and size exclusion remove aggregates or leftover host proteins that affinity capture missed. They are requested when the assay is sensitive to those species, not automatically on every milligram project.