Affinity chromatography is a method of separating biochemical mixtures based on
a highly specific interaction such as that between antigen and antibody,
enzyme and substrate, or receptor and ligand.
Uses Affinity chromatography can be used to:
Purify and concentrate a substance from a mixture into a buffering solution
Reduce the amount of a substance in a mixture
Discern what biological compounds bind to a particular substance
Purify and concentrate an enzyme solution.
Principle The stationary phase is typically a gel
matrix, often of agarose; a linear sugar molecule derived from algae. Usually the
starting point is an undefined heterogeneous group of molecules in
solution, such as a cell lysate, growth medium or blood serum. The molecule of
interest will have a well known and defined property, and can be exploited
during the affinity purification process. The process itself can be
thought of as an entrapment, with the target molecule becoming trapped on a
solid or stationary phase or medium. The other molecules in the mobile phase will
not become trapped as they do not possess this property. The stationary
phase can then be removed from the mixture, washed and the target molecule
released from the entrapment in a process known as elution. Possibly the
most common use of affinity chromatography is for the purification
of recombinant proteins. Batch and column setup
Binding to the solid phase may be achieved by column chromatography
whereby the solid medium is packed onto a column, the initial mixture run
through the column to allow setting, a wash buffer run through the column and
the elution buffer subsequently applied to the column and collected. These steps
are usually done at ambient pressure. Alternatively, binding may be achieved
using a batch treatment, for example, by adding the initial mixture to the solid
phase in a vessel, mixing, separating the solid phase, removing the liquid
phase, washing, re-centrifuging, adding the elution buffer, re-centrifuging and
removing the eluate. Sometimes a hybrid method is employed
such that the binding is done by the batch method, but the solid phase with
the target molecule bound is packed onto a column and washing and elution are
done on the column. A third method, expanded bed adsorption,
which combines the advantages of the two methods mentioned above, has also been
developed. The solid phase particles are placed in a column where liquid phase is
pumped in from the bottom and exits at the top. The gravity of the particles
ensure that the solid phase does not exit the column with the liquid phase.
Affinity columns can be eluted by changing salt concentrations, pH, pI,
charge and ionic strength directly or through a gradient to resolve the
particles of interest. Specific uses
Affinity chromatography can be used in a number of applications, including
nucleic acid purification, protein purification from cell free extracts,
and purification from blood. = Immunoaffinity=
Another use for the procedure is the affinity purification of antibodies from
blood serum. If serum is known to contain antibodies against a specific
antigen then it can be used for the affinity purification of that antigen.
This is also known as Immunoaffinity Chromatography. For example if an
organism is immunised against a GST-fusion protein it will produce
antibodies against the fusion-protein, and possibly antibodies against the GST
tag as well. The protein can then be covalently coupled to a solid support
such as agarose and used as an affinity ligand in purifications of antibody from
immune serum. For thoroughness the GST protein and the
GST-fusion protein can each be coupled separately. The serum is initially
allowed to bind to the GST affinity matrix. This will remove antibodies
against the GST part of the fusion protein. The serum is then separated
from the solid support and allowed to bind to the GST-fusion protein matrix.
This allows any antibodies that recognize the antigen to be captured on
the solid support. Elution of the antibodies of interest is most often
achieved using a low pH buffer such as glycine pH 2.8. The eluate is collected
into a neutral tris or phosphate buffer, to neutralize the low pH elution buffer
and halt any degradation of the antibody's activity. This is a nice
example as affinity purification is used to purify the initial GST-fusion
protein, to remove the undesirable anti-GST antibodies from the serum and
to purify the target antibody. A simplified strategy is often employed
to purify antibodies generated against peptide antigens. When the peptide
antigens are produced synthetically, a terminal cysteine residue is added at
either the N- or C-terminus of the peptide. This cysteine residue contains
a sulfhydryl functional group which allows the peptide to be easily
conjugated to a carrier protein). The same cysteine-containing peptide is also
immobilized onto an agarose resin through the cysteine residue and is then
used to purify the antibody. Most monoclonal antibodies have been
purified using affinity chromatography based on immunoglobulin-specific Protein
A or Protein G, derived from bacteria. = Immobilized metal ion affinity
chromatography= Immobilized metal ion affinity
chromatography is based on the specific coordinate covalent bond of amino acids,
particularly histidine, to metals. This technique works by allowing proteins
with an affinity for metal ions to be retained in a column containing
immobilized metal ions, such as cobalt, nickel, copper for the purification of
histidine containing proteins or peptides, iron, zinc or gallium for the
purification of phosphorylated proteins or peptides. Many naturally occurring
proteins do not have an affinity for metal ions, therefore recombinant DNA
technology can be used to introduce such a protein tag into the relevant gene.
Methods used to elute the protein of interest include changing the pH, or
adding a competitive molecule, such as imidazole.
= Recombinant proteins= Possibly the most common use of affinity
chromatography is for the purification of recombinant proteins. Proteins with a
known affinity are protein tagged in order to aid their purification. The
protein may have been genetically modified so as to allow it to be
selected for affinity binding; this is known as a fusion protein. Tags include
glutathione-S-transferase, hexahistidine, and maltose binding
protein. Histidine tags have an affinity for nickel or cobalt ions which have
been immobilized by forming coordinate covalent bonds with a chelator
incorporated in the stationary phase. For elution, an excess amount of a
compound able to act as a metal ion ligand, such as imidazole, is used. GST
has an affinity for glutathione which is commercially available immobilized as
glutathione agarose. During elution, excess glutathione is used to displace
the tagged protein. = Lectins=
Lectin affinity chromatography is a form of affinity chromatography where lectins
are used to separate components within the sample. Lectins, such as
Concanavalin A are proteins which can bind specific carbohydrate molecules.
The most common application is to separate glycoproteins from
non-glycosylated proteins, or one glycoform from another glycoform.
See also Weak Affinity Chromatography
References External links
[1], Affinity Chromatography Handbook by GE Healthcare Life Sciences