Column Chromatography Resource
The following resource is intended to elaborate on the material covered in lecture, refer back to this supplement as needed to review details of
column chromatography techniques in protein purification schemes.
Glossary
Lysate: the starting material that includes the protein of interest and other soluble proteins and cellular components in solution as a result of
breaking down the membranes of cells
Column: a glass or plastic tube that is packed with a solid material (beads/resin)
Beads/resin: the solid materials that stay within the column during purification
Flowthrough: the buffer solution and any proteins from your lysate that pass through the column without binding
Wash: the process in which buffer runs through the column to remove any unbound or weakly bead-bound proteins in ion-exchange and affinity
chromatography
Elution: the process in which buffer runs through the column to remove the protein(s) of interest off the column
Eluates: proteins that come off the column during elution
Fractions: a specific volume that comes off the column and is collected during the elution process (and often during washes)
Pool: the process of combining collected fractions to increase the yield of the protein of interest
Generalized Example Protocols:
There is great variation in the equipment and materials used for different methods of column chromatography, and many laboratories have
automated systems to perform these protocols. However, there are general steps in common regardless of the type of chromatography or system
used. Here we outline the generalized steps for size-exclusion chromatography (SEC), ion-exchange chromatography (IEC), and affinity
chromatography and highlight the specific differences between the three techniques for each step.
Pack a column with beads and equilibrate the packed column.
SEC IEC Affinity
Beads contain: pores of defined size (fractionation Beads contain: covalently attached functional groups Beads contain: covalently attached ligand (like
range) that are either positively (anion exchange) or an antibody or binding partner) to which the
Equilibration: Buffer fills the space inside the pores negatively (cation exchange) charged protein of interest binds
and between the beads. The buffer remains the same Equilibration: Buffer ensures that all of the charged Equilibration: Buffer ensures proper binding
throughout the SEC protocol. functional groups are bound to exchangeable ions, between ligand and protein of interest.
such as chloride or sodium.
Load the soluble lysate onto the column.
SEC IEC Affinity
Exclusion: Large proteins do not enter the porous beads, whereas Binding: Charged proteins replace the Binding: Proteins with ligand affinity
some intermediate proteins and small proteins do. exchangeable ions bound to the beads. bind to the column.
Pass several washes of buffer through the column to wash off any unbound or weakly bound proteins.
SEC IEC Affinity
NONE: There are NO Wash flowthrough: proteins with the same charge as the Wash flowthrough: proteins that do not have affinity for the ligand
washes in SEC. resin or zero charge
Elute proteins off the column.
SEC IEC Affinity
Elution (or running) Elution buffer: Modifications of either buffer (1) salt Elution buffer: Modifications in buffer composition weaken
buffer: moves sample concentration or (2) pH weaken column-protein interactions. binding affinity. Most commonly, (1) pH changes alter
through the column (1) Increases in salt concentration gradually cause salt ions to protein and/or ligand charge, (2) increases in salt
Eluates: Large proteins compete with the bound protein for space on the column, or concentration reduce electrostatic interactions, or (3)
come off the column first, (2) changes in pH titrate the charged groups on the column inclusion of a molecule that binds either the protein or ligand
followed by intermediate, and/or the bound proteins until they are neutral or of opposite replaces the column-protein interaction.
and then small proteins. charges. Eluates: Proteins that were bound to the ligand elute.
Eluates: (1) Proteins with a lower net charge elute first, as
they are easier to displace, and proteins with a greater net
charge elute later. (2) Decreases in pH with an anion
exchanger and increases in pH with a cation exchanger cause
bound proteins to elute.
Collect and monitor fractions in small volumes as eluates come off the column.
All chromatographic techniques require monitoring of the collected fractions for the protein of interest, as fractions without the protein of
interest are discarded and some fractions with the protein of interest are pooled. Monitoring can be done by measuring absorbance at 280 nm,
running an SDS-PAGE gel, assaying fractions for activity if an assay is available, or a combination of these methods. Some systems have some
automated monitoring, while others are done manually.
A B C
Example output from different methods of monitoring column chromatography purification schemes. (A) Monitoring absorbance at 280nm is a
general technique that will give information about total protein amount. (B) SDS-PAGE gives more information about each fraction including
protein molecular weight and abundance. (C) In some cases (e.g., enzyme purification), activity can be assayed from the fractions to determine if
the protein purified is functional.
