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BCH5425 Molecular Biology and Biotechnology

BCH5425 Molecular Biology and Biotechnology
Spring 1998
Dr. Michael Blaber 

Lecture 31

Protein Purification: Gel Filtration, Affinity and Hydrophobic resins; Preparation of Resin, Plumbing

Gel filtration

Gel filtration does not rely on any chemical interaction with the protein, rather it is based on a physical property of the protein - that being the effective molecular radius (which relates to mass for most typical globular proteins).

Where will a protein elute in a gel filtration experiment?

As a general rule of thumb, the excluded volume (Vo) is approximately equal to one third of the column volume, the included volume is approximately equal to two thirds of the column volume

Affinity chromatography

Affinity chromatography is a general term which applies to a wide range of chromatographic media. It can be basically thought of as some inert resin to which has been attached some compound which has a specific affinity for your protein of interest.

In each case, the type of resins used and the method of attachment may vary, as will the method of elution. One generalization regarding method of elution is that the bound ligand can be competed off of the column's functional group by including in the elution buffer a high concentration of the free functional group. For example, if the functional group of the column is a cofactor, then the bound protein can be competed off the column by passing a buffer containing a high concentration of cofactor (or cofactor analog) through the column.

Other methods of elution include changing the buffer conditions such that the protein is no longer in the native state (since it is the native state which confers the structure required for the specific binding interaction). This can be achieved by changing pH or by adding denaturing agents such as urea or guanidine.

With affinity chromatography, typically the purification achieved in a single step can be dramatic - on the order of several thousand fold. Single step purifications with specific affinity columns are not unheard - in fact it is an ideal goal of purification - a matrix which recognizes only the protein of interest and none other.

Hydrophobic resins

Hydrophobic resins contain a non-polar functional group, such as an alkane or aromatic group.

 Due to the nature of hydrophobic interactions and ionic strength, hydrophobic chromatography and ion exchange chromatography can be conveniently used sequentially. For example, after ion exchange the protein is in high salt conditions, thus it can be loaded directly onto a hydrophobic column. Conversely, a hydrophobic column is eluted in low salt, which is a requirement for binding to an ion exchange resin.

A distinction should be noted between hydrophobic interaction chromatorgraphy and reverse phase chromatography

Preparation of resins

The steps in preparing a chromatographic resin typically involve:

  1. Hydration of resin
  2. Decanting fines
  3. Equilibrating the resin and preparing a slurry
  4. Degassing the slurry

Packing the column

Low pressure columns are typically packed using gravity.


Chromatography systems may be run using only gravity and a beaker to collect the appropriate fraction. Most common systems, however, will include the following:

Note that the bottom of the safety loop is lower than the outlet to the fraction collector.

1998 Dr. Michael Blaber