Introduction
- Page ID
- 73053
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\(\newcommand{\avec}{\mathbf a}\) \(\newcommand{\bvec}{\mathbf b}\) \(\newcommand{\cvec}{\mathbf c}\) \(\newcommand{\dvec}{\mathbf d}\) \(\newcommand{\dtil}{\widetilde{\mathbf d}}\) \(\newcommand{\evec}{\mathbf e}\) \(\newcommand{\fvec}{\mathbf f}\) \(\newcommand{\nvec}{\mathbf n}\) \(\newcommand{\pvec}{\mathbf p}\) \(\newcommand{\qvec}{\mathbf q}\) \(\newcommand{\svec}{\mathbf s}\) \(\newcommand{\tvec}{\mathbf t}\) \(\newcommand{\uvec}{\mathbf u}\) \(\newcommand{\vvec}{\mathbf v}\) \(\newcommand{\wvec}{\mathbf w}\) \(\newcommand{\xvec}{\mathbf x}\) \(\newcommand{\yvec}{\mathbf y}\) \(\newcommand{\zvec}{\mathbf z}\) \(\newcommand{\rvec}{\mathbf r}\) \(\newcommand{\mvec}{\mathbf m}\) \(\newcommand{\zerovec}{\mathbf 0}\) \(\newcommand{\onevec}{\mathbf 1}\) \(\newcommand{\real}{\mathbb R}\) \(\newcommand{\twovec}[2]{\left[\begin{array}{r}#1 \\ #2 \end{array}\right]}\) \(\newcommand{\ctwovec}[2]{\left[\begin{array}{c}#1 \\ #2 \end{array}\right]}\) \(\newcommand{\threevec}[3]{\left[\begin{array}{r}#1 \\ #2 \\ #3 \end{array}\right]}\) \(\newcommand{\cthreevec}[3]{\left[\begin{array}{c}#1 \\ #2 \\ #3 \end{array}\right]}\) \(\newcommand{\fourvec}[4]{\left[\begin{array}{r}#1 \\ #2 \\ #3 \\ #4 \end{array}\right]}\) \(\newcommand{\cfourvec}[4]{\left[\begin{array}{c}#1 \\ #2 \\ #3 \\ #4 \end{array}\right]}\) \(\newcommand{\fivevec}[5]{\left[\begin{array}{r}#1 \\ #2 \\ #3 \\ #4 \\ #5 \\ \end{array}\right]}\) \(\newcommand{\cfivevec}[5]{\left[\begin{array}{c}#1 \\ #2 \\ #3 \\ #4 \\ #5 \\ \end{array}\right]}\) \(\newcommand{\mattwo}[4]{\left[\begin{array}{rr}#1 \amp #2 \\ #3 \amp #4 \\ \end{array}\right]}\) \(\newcommand{\laspan}[1]{\text{Span}\{#1\}}\) \(\newcommand{\bcal}{\cal B}\) \(\newcommand{\ccal}{\cal C}\) \(\newcommand{\scal}{\cal S}\) \(\newcommand{\wcal}{\cal W}\) \(\newcommand{\ecal}{\cal E}\) \(\newcommand{\coords}[2]{\left\{#1\right\}_{#2}}\) \(\newcommand{\gray}[1]{\color{gray}{#1}}\) \(\newcommand{\lgray}[1]{\color{lightgray}{#1}}\) \(\newcommand{\rank}{\operatorname{rank}}\) \(\newcommand{\row}{\text{Row}}\) \(\newcommand{\col}{\text{Col}}\) \(\renewcommand{\row}{\text{Row}}\) \(\newcommand{\nul}{\text{Nul}}\) \(\newcommand{\var}{\text{Var}}\) \(\newcommand{\corr}{\text{corr}}\) \(\newcommand{\len}[1]{\left|#1\right|}\) \(\newcommand{\bbar}{\overline{\bvec}}\) \(\newcommand{\bhat}{\widehat{\bvec}}\) \(\newcommand{\bperp}{\bvec^\perp}\) \(\newcommand{\xhat}{\widehat{\xvec}}\) \(\newcommand{\vhat}{\widehat{\vvec}}\) \(\newcommand{\uhat}{\widehat{\uvec}}\) \(\newcommand{\what}{\widehat{\wvec}}\) \(\newcommand{\Sighat}{\widehat{\Sigma}}\) \(\newcommand{\lt}{<}\) \(\newcommand{\gt}{>}\) \(\newcommand{\amp}{&}\) \(\definecolor{fillinmathshade}{gray}{0.9}\)This module provides an introduction to Affinity Chromatography separations. This is a technique commonly used to purify a biomolecule of interest based on the specific interaction of the biomolecule with a ligand immobilized on a stationary phase. Affinity chromatography is widely used in biotechnology research and in industry. Using this technique, molecules of interest can be purified in a one-step separation with high purity and high recovery. The purification of the molecule does not require harsh conditions and hence is preferred for biomolecule separation. Affinity chromatography has also found applications in studying drug/ligand binding to proteins of interest. This technique is useful in the purification of peptides, chiral molecules, antibodies, nucleic acids, and proteins.
The aim of this module is to present the basic theory and applications of affinity chromatography. This module is meant to be utilized in the classroom as a teaching resource or by anyone interested in learning about affinity chromatography. As a teaching resource, the module can be used directly, or the students can be given an in-class problem set, which will lead to further discussion on affinity chromatography.
Objective
The students will be able to describe the principles of affinity chromatography. They will be able to apply and evaluate the technique of affinity chromatography to the purification of a biomolecule of interest.
Goals:
Theory and applications of affinity chromatography will be described with emphasis on the following:
- Devising a method of purification for a biomolecule of interest
- Steps involved in affinity chromatography purification
- Methods for evaluating purity of the biomolecule of interest
- Broader applications of the technique
Links are provided for additional reading material in the field of affinity chromatography.
History
Although the word affinity chromatography was coined in the 1960s, and has since been recognized as a powerful tool for bio-specific purification, the bio-specific binding was first used in 1910 by the German Pharmacologist Emil Starkenstein. He demonstrated specific binding of the enzyme alpha-amylase to starch as a means for purification of the enzyme. Subsequent studies reported the use of insoluble affinity material as both the stationary phase and the support material.
The method was further developed by Prof. Pedro Cuatrecasas from the University of California San Diego and Prof. Meir Wilchek from Weizmann institute of Science in Israel in 1968. Their method was developed to purify the common enzymes staphylococcal nuclease, a-chymotrypsin, and carboxypeptidase A using sepharose-enzyme inhibitor as the matrix. You can read more about their discovery in the following paper:
- Cuatrecasas P, Wilchek H, and Anfinsen CB (1968). "Selective enzyme purification by affinity chromatography". Proc. Nat. Acad. Sci. USA61: 636-43.
The scientists were awarded the Wolf Prize in Medicine in 1987 for their work in Affinity Chromatography.
