Chromatography Cheat Sheet

Chromatography Chromatographic methods Chromatographic methods involve a column of an insoluble material that can bind molecules based on specific properties common to proteins The solution containing the mixture of proteins is then allowed to pass through the column; the protein of interest may bind (depending on its properties), while at least some impurities remain in solution and leave the column Ion exchange chromatography Molecules vary in their charge properties and will interact differently with charged chromatography media based on variations in their overall charge, charge density, and surface charge distribution The pH at which the net charge on a protein becomes zero is termed the isoelectric point (pI) of that protein. If a buffer has a pH greater than the pI of the protein of interest, the protein will have a net negative charge Negatively charged protein Negatively charged cation-exchange resin Positively charged protein Positive charged anion-exchange resin The basic steps of ion exchange chromatography Equilibration The purpose of this step is to set up the column for sample binding, so the chosen buffer should have all the necessary conditions for sample binding Column loading Load the sample in the column, ensuring it has the same conditions as the binding buffer selected so it will bind to the column Washing Wash the column to elute contaminating proteins or proteins that are not bound to the column Gradient elation Gradually increase the salt concentration within the column Wash & re-equilibration Complete the process by washing the column once again with a high salt solution to remove any remaining molecules bound to the column and begin re-equilibration to perform another purification