Affinity Chromatography

Lehrstuhl für Siedlungswasserwirtschaft
Ingenieurfakultät Bau Geo Umwelt

Technische Universität München
Affinity Chromatography
PD Dr. J. Graßmann; PD Dr. T. Letzel

Applications
• Based on highly specific interactions between a molecule of interest (= target analyte) an a
immobilized molecule (= ligand), which specifically binds and „catches“ the analyte.
• AC can therefore be used to purify a particular molecule, which was originally solved in a
complex sample matrix (e.g. cell extract).
• AC also provides the possibility to concentrate an analyte of interest and simultaneously get
rid of the sample matrix, which is e.g. particularly helpful for the purification and concentration
of enzyme solutions.
• You may also use AC to identify molecules, which bind to an e.g. antibody or receptor of
interest or to a particular substance in general
• Study of receptor-drug interaction
• Study of interactions between enzyme and inhibitor, receptor and receptor ligands, etc.

Procedure
• Starting point is a mixture which contains the target analyte (e.g. blood serum or urine)
• Affinity medium is equilibrated in binding buffer
• The sample is then introduced to a stationary phase (e.g. agarose gel)
• Conditions favor specific binding of the target molecule to a complementary substance (the
ligand). Target substances bind specifically, but reversibly. Unbound material (i.e. sample
matrix) washes through the column.
• The analyte is then eluted from the stationary phase and collected, which results in a highly
purified and concentrated analyte solution.
• Affinity medium is re-equilibrated in binding buffer.

Procedure - Example (with an antibody as ligand)
Column with stationary phase e.g. agarose gel
and immobilized ligand e.g. antibodies
Analyte specifically binds

the immobilized antibody ligands
Addition of sample Washing to clean

with analyte the sample of Elution of analyte
and matrix matrix compounds

Specific Elution
Elution of analyte can be done specifically by adding a competitive
ligand (here).

Specific Elution
Option 1 Option 2
Elution by adding a Elution by adding a
molecule, which molecule, which
specifically binds to the specifically binds to the
ligand to release the analyte to disrupt the
analyte. interaction with the ligand

Non-specific Elution
Elution can also be done non-specifically by changing the pH value, the ionic strength, the
temperature or the polarity. All of these lower the KD value, which results in the release of the
analyte.
pH  the ionization of charged groups, either on the ligand or the (protein-)analyte is altered by
pH changes (compare chapter „Polarity and Solubility“). Those alteration cause changes in
affinity of the binding site or may alter the analyte conformation, both of which causing the
disruption of the ligand-analyte interaction.
Ionic strength  alters the interaction between ligand and (protein-)analyte. An increasing ionic
strength using NaCl is typically applied to elute the target compound(s).
Solvent polarity  decreasing polarity of the solvent is usually a good means to disrupt the
interaction between analyte and ligand.
Chaotropic eluent  Those can alter the conformation of a protein-analyte. However this is often
not recommended due to the denaturation of the protein target.

Step elution (e.g. with NaCl)
Binding
Elution
conditions
Time

Gradient elution (e.g. with NaCl)
Elution
Analyte 2
Elution
Binding Analyte 1
conditions
Time

Typical Applications
Ligand Target analyte
Antibody Antigen (e.g. virus, protein)
Antigen Antibody
Enzyme (e.g. glutathione s-transferase) Substrate (e.g. glutathione), inhibitor, cofactor
Cofactor, substrate, inhibitor Enzyme
Nucleic acid Primer, i.e. complementary sequence, nucleic acid binding proteins
Primer, i.e. complementary sequence, nucleic Nucleic acid
acid binding proteins
Receptor Receptor ligand (e.g. Hormones, vitamins)
Receptor ligand Receptor
Metal ions Proteins tagged with histidine, cysteine or tryptophan residues
Lectin Carbohydrates, (e.g. on cell surfaces or viruses)
Carbohydrates Lectin
Ligand-Protein complex Another protein (protein-protein interaction  „pull down assay“)
Ligand and analyte are often interchangable as long as the requirements are fulfilled. E.g. an enzyme may
serve as ligand to capture an enzymatic substrate. However also the substrate can serve as ligand to capture
an enzyme.
Applicability from two perspectives

Common terms
Matrix: for ligand attachment. Matrix should be

chemically and physically inert.
Spacer arm: used to improve binding between
Y
ligand and target molecule by overcoming any
effects of steric hindrance.
Ligand: molecule that binds reversibly to a
V
specific target molecule or group of target
molecules.

Requirements
• Availability of a ligand which binds the analyte of interest

• Ligand can be covalently attached to the stationary phase matrix
• Ligand has to be stable and maintain its binding affinity
• Ligand needs to be unspecific for sample matrix components
• Binding between ligand and analyte has to be reversible, so the analyte can be
eluted from the ligand.
• The analyte needs to remain intact during the binding to and removal from the
ligand

Immobilized-metal affinity chromatography (IMAC)
• IMAC is based on the affinity of transition metal ions such as Zn2+, Cu2+, Ni2+ and
Co2+ to histidine and cysteine
• Proteins and peptides that have an affinity for metal ions can be separated by using
metal chelate affinity chromatography
• The metals are immobilized onto a chromatographic medium by chelation
• Certain amino acids, e.g. histidine and cysteine, form complexes with the chelated
metals

Immobilized-metal affinity chromatography (IMAC)
Elution is e.g. specifically done with an
excess amout of imidazole, which acts as
a competitor and thus disrupts the
interaction between His-tag and Ni2+
Imidazole
Drawing of chemical structures was performed with „MarvinSketch 14.8.25.0“, ChemAxon (www.chemaxon.com)

Further possibilities to purify analytes by means of AC
• Avidin/Streptavidin-Biotin interaction  the protein to be purified is tagged with biotin. It

specifically binds to the protein Avidin/Streptavidin, which serves as ligand.
• Glutathione s-transferase (GST) or proteins tagged with GST can be captured by immobilized
glutathione, which serves as ligand.
• Immobilized lectin is used to purify glycoproteins, gylcolipids or polysaccharides due to lectin
interacting with certain sugar residues.
• Pull down assay uses one protein (=bait protein) to capture a second protein (=prey protein). It
can be used e.g. for the screening and identification of unknown protein-protein interactions.

Pull down assay
Elution of the bait-prey protein complex
e.g. SDS PAGE (compare Bioanalytics slides) to determine molecular weight

of prey protein  further identification e.g. via trypsin digestion and determination of
peptides MW via mass spectrometry  data base search
Advantages of Affinity chromatography
• Extremely high specificity

• High degrees of purity can be obtained
• The process is highly reproducible
• Binding sites of biological molecules can be investigated

Affinity Chromatography

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Lehrstuhl für Siedlungswasserwirtschaft

Ingenieurfakultät Bau Geo Umwelt

PD Dr. J. Graßmann; PD Dr. T. Letzel

PD Dr. J. Graßmann; PD Dr. T. Letzel

PD Dr. J. Graßmann; PD Dr. T. Letzel

Analyte specifically binds

Addition of sample Washing to clean

PD Dr. J. Graßmann; PD Dr. T. Letzel

PD Dr. J. Graßmann; PD Dr. T. Letzel

PD Dr. J. Graßmann; PD Dr. T. Letzel

PD Dr. J. Graßmann; PD Dr. T. Letzel

Step elution (e.g. with NaCl)

PD Dr. J. Graßmann; PD Dr. T. Letzel

PD Dr. J. Graßmann; PD Dr. T. Letzel

PD Dr. J. Graßmann; PD Dr. T. Letzel

Matrix: for ligand attachment. Matrix should be

Spacer arm: used to improve binding between

Ligand: molecule that binds reversibly to a

PD Dr. J. Graßmann; PD Dr. T. Letzel

• Availability of a ligand which binds the analyte of interest

PD Dr. J. Graßmann; PD Dr. T. Letzel

PD Dr. J. Graßmann; PD Dr. T. Letzel

PD Dr. J. Graßmann; PD Dr. T. Letzel

• Avidin/Streptavidin-Biotin interaction  the protein to be purified is tagged with biotin. It

PD Dr. J. Graßmann; PD Dr. T. Letzel

Elution of the bait-prey protein complex

e.g. SDS PAGE (compare Bioanalytics slides) to determine molecular weight

• Extremely high specificity

PD Dr. J. Graßmann; PD Dr. T. Letzel

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