This blog post about pH in the context of protein purification is the third in a series about optimizing conditions for protein purification. Although pH is most commonly seen as an issue in ion-exchange chromatography, it also plays a critical role in affinity tag protein purification.
Using denaturing conditions is a way to coax insoluble proteins into solution by reducing hydrophobic effects and unfolding the aggregates. Denaturing agents are also useful for testing protein folding dynamics, protein elution from a column, and regenerating a resin column.
When conducting affinity purification of a protein, there are many variables to consider in order to optimize purity and yield. One such condition to control is high salt loading, which decreases impurities in the final product.
Protein fusion tags make affinity chromatography possible for many proteins, particularly those for which no affinity-quality antibody is available. In addition to improving conditions for purification, tags can facilitate detection and protein interaction studies.
Affinity purification is a powerful method for isolating proteins. In addition to providing affinity to separate proteins from solution, tags can have a profound impact on a protein’s folding or activity.