Biology Reference
In-Depth Information
Antibody-Affinity Chromatography
Another important type of affinity chromatography employs columns with attached
antibodies
[32,38]
. Antibody-affinity chromatography is more versatile and powerful than
lectin-affinity chromatography. While use of lectins is limited to membranes possessing
surface carbohydrates, appropriate antibodies can bind to any surface antigen including
carbohydrates and proteins. Lipids and nucleic acids are antigenic only when bound
to carbohydrates or proteins. In addition, antigen-antibody binding is much stronger than
carbohydrate-lectin binding.
The term antigen was coined in 1899 by Hungarian microbiologist Ladislas Deutsch (he
also had an alias, Laszio Detre). Antigen is a contraction of the original name 'antisomato-
gen'. An antigen is simply any molecule or part of a molecule that is recognized by the
immune system and can bind to the antigen-binding site of an antibody. Antibodies have
the ability to recognize and bind specifically to an antigen in the presence of a vast solution
of molecules that are often similar in structure. The precise specificity of an antibody for an
antigen makes them ideal for membrane purification by affinity chromatography. Countless
custom antibodies are now commercially available for attachment to column matrix material.
A crude membrane homogenate is passed over the antibody column whereupon only the
membrane fractions containing the appropriate surface antigen bind. Other membrane frac-
tions pass through the column and are discarded. After washing the column, the bound
membrane is released by altering the pH of the washing solution.
Ligand-Receptor Affinity Chromatography
Another type of affinity chromatography employs ligand-receptors. The outer surface of
the plasma membrane has a large number of proteinaceous receptors whose function is to
bind specifically to external molecules referred to as ligands. Included in a long list of ligands
are small signaling peptides, hormones, neurotransmitters, vitamins, and toxins. An unusual
application is employing zinc-affinity columns to isolate proteins that require zinc cofactors
to function
[39]
. Ligands bind to, and dissociate from receptors according to the law of mass
action. By this methodology, ligands are covalently bound to the column matrix and the cell
homogenate is passed over the column. Membranes with the receptor for the ligand bind to
the column and the non-bound membranes pass through the column and are discarded.
Ligand-bound membrane fractions are released from the column by adding a large excess
of free ligand. While ligand affinity columns have occasionally been used to separate
membrane fractions, their major use has been in isolating and purifying receptors for signal
transduction studies.
Anion Exchange Chromatography
Ion exchange chromatography
[40]
has been an important scientific tool since its discovery
in about 1850. Anion exchange columns are made of resin beads, often agarose or cellulose, to
which is covalently bound a cationic functional group. The stationary phase (the resin beads)
interacts with anions dissolved in a mobile, aqueous phase. Anion exchange chromatography
separates compounds based on their net negative surface charge. Since biological membranes
