Affinity Chromatography

Purification via affinity chromatography is a powerful technique to separate your protein of interest from your sample. The proteins are marked with "tags" (Fig. 3, Table 4) which give them specific binding properties. A suitable immobilized ligand binds to the tagged protein (Fig. 1). So your protein of interest is separated from the rest of your sample.

Principles of Affinity Chromatography

In general affinity chromatography is composed of a stationary phase (solid phase) and a mobile phase (Fig. 1). The mobile phase is your cell lysate or any mixture that contains biomolecules. A ligand that binds the target molecule is attached covalently to the solid phase. The interaction between the solid and the mobile phase are exploited by affinity chromatography to get your desired substance in a pure form. The target molecule binds to the ligand, whereas most of the other molecules flow through. The target biomolecule is eluted by changing conditions (pH or salt concentrations) or by competition with a free ligand.The most important property which the solid phase should have is ligand immobilization. Various materials like acrylates or silica gels are appropriate. To prevent steric interference of the target molecule to the ligand, an inhibitor is attached to the solid phase. This inhibitor is known as the spacer (Fig. 2). The classic spacer is an inhibitor containing a hydrocarbon chain (CH2 spacer). Chemicals like cyanogen bromide or epoxide can functionalize the solid phase with hydrocarbon chains which result in various carbon chain lengths depending on the chemical (Table 2).

Stationary and mobile phase bound to each other

Workflow of an affinity chromatography using a drip column

The stationary Phase

The stationary phase of an affinity chromatography, also called the solid phase, consists of the Core, the Spacer and the Ligand and on occasion a metal ion that is coupled to the ligand (e.g. His-tag purification).

The spacer

The spacer of an affinity bead can vary in size and length. Longer chain lengths have a higher mobility and are thus crucial for stericaly hindered tags.

Chemical	Chain
Cyanogen bromide	C1
Epoxide	C3
Epoxide with C6 acid	C10
Diamin	C10

The core

Bead material	Features
Agarose	hydrophilic almost no unspecific bonds the gold standard for protein purification
Silica Gel	nanoporous (leads to unspecific bonds) functionalized via silane silanes are washed away by alkaline buffer reduced stability applications: bound nucleic acids chaotropic
Aluminium oxide	acidic surface binds amines irreversibly used to reduce the amount of specific substances
Acrylate	partially hydrophobic (unspecific binding possible) monodisperse particles used for cell separation
Organic polymers	partially hydrophobic (unspecific bounds possible) monodisperse particles can be used for ligand coupling not recommended for protein purification because of unspecific bounds

Advantages of porous agarose

easily dispensed to fill and "pack" columns with resin beds of any size
large enough that biomolecules (proteins, etc.) can flow as freely into and through the beads
Ligands are covalently attached to the bead polymer (external and internal surfaces) by various means
Beaded agarose is good for routine applications as it allows a setup, making it suitable for gravity-flow, low-speed-centrifugation, and low-pressure procedures

The ligand

The ligand is responsible for the affinity of the bead. In some cases the affinity of the ligand can be further modified. A noteworthy example here are His-tag purifications. In this case the ligand is a chelator coupled with a metal ion. The metal ion is responsible for the balance between affinity and specificity of the purification.

Ligand	Target
Antibody	Antigen
Iron-, aluminium ions	Phosphoproteins
Avidin	Biotin
Glutathione	His-tagged proteins

The mobile/liquid phase

Affinity chromatography relies on the presence of an affinity tag - typically a peptide or protein sequence which can be added to the protein of interest on the DNA level. For small tags, this can be done by a two-step PCR, but in most cases dedicated cloning vectors are available. Once equipped with a suitable affinity tag, the gene of interest is expressed and the recombinant protein carries the respective additional amino acids.

Affinity chromatography resins or matrices are typically agarose or magnetic agarose beads that are covalently coupled to a molecule that specifically binds to the affinity tag. There is a great variety in tag-resin partner chemistries and interaction types. See table 3 for a first overview.

Stylized protein with N- and C-terminal ends

Table 4 on the other hand lists the most commonly used affinity tags and their individual features

Tag	Amino acid sequence	DNA Sequence	Size	Specificity of interaction (KD)	Protein Yield per ml resin	Elution conditions
Poly His-tag	6-14 Histidine residues	CAT CAC CAT CAC CAT CAC	840.8-1,937.9 Dalton	10 µM	up to 80 mg/ml (1)	Imidazole or histidine, or at low pH
GST-tag	GST-tag protein (whole protein)	Plasmid / Basic cloning vector	26 kDa protein	1 µM	10-12 mg/ml	Reduced glutathione
Strep®-tag	WSHPQFEK	TGG TCG CAT CCG CAG TTC GAG AAG	1,058.1 Dalton	300 nM	up to 9 mg/ml	Desthiobiotin or biotin
Rho1D4 / 1D4-tag	TETSQVAPA	ACC GAG ACT TCC CAG GTG GCG CCA GCT	902.9 Dalton	20 nM (4)	3-4 mg/ml	Rho1D4 peptide, low pH, or protease digest
FLAG®-tag	DYKDDDDK	GAC TAC AAA GAC GAT GAC GAC AAG	1,012.9 Dalton	100 nM	0.6-1 mg/ml	DYKDDDDK peptide

Bind-Wash-Elute: The three steps of Affinity Purification

Most affinity purification protocols follow the same three steps:

1. Binding:
A complex solution containing the tagged protein is applied to the column and binds based on the affinity tag - matrix interaction

2. Washing:
Other proteins which bind unspecifically are washed away with suitable buffers

3. Elution:
Specifically bound protein is eluted from the column, typically by competitive binding of a similar molecule (e.g. histidine and imidazole), by cutting off the tag with a protease or by destabilization of the affinity tag - matrix interaction e.g. by a change of pH

Affinity tag applications

While the term "affinity chromatography" implies that the protein of interest is being purified via the affinity tag, there are a number of applications that can be done in addition to purificaton. These include:

Detection: Specific antibodies are available for most affinity tags, so that tagged proteins can be detected in Western Blots, via immunostaining, in ELISA assays or other antibody-based applications.

Immobilization: Affinity tags can be used to immobilize tagged proteins, e.g. on surface plasmon resonance chips, on ELISA plates or other surfaces. The immobilized proteins can then be assessed e.g. for their ligand binding kinetics.

Pulldown: Affinity-tagged proteins can be pulled down from complex solutions e.g. via affinity magnetic beads. They can also be immobilized via affinity beads and used to pull down interaction partners from complex mixtures, such as cell lysates.

Affinity chromatography methods in the lab

Magnetic Beads

Magnetic Beads Ligands bound to magnetic beads are a fast and smart way to purify proteins (Read the complete guide here). To perform the purification you need a magnetic bead separator which purifies your protein of interest. Our scientist Roland Fabis PhD made a complete MagBead tutorial on YouTube (You can watch it here ).

Glas flask with thin tube as part of an FPLC

FPLC - Fast protein liquid chromatography

FPLC is the standard method for protein purification via affinity chromatography. The advantage of FPLC is that the buffer flow rate is controlled by a positive-displacement pump and the total flow rate of the buffer is kept constant. So FPLC is very suitable for method development and the results are reproducible. The used pressure is with typically less than 5 bar relativley low. The drawback of the method is that you need to buy a very expensive chromatography system.

Drip columns

With drip columns you can work directly at your bench. They are small disposable plastic columns packed with chromatography affinity resin. In contrast to the FPLC method you cannot control pressure and flow rate.

Batch spin

The batch spin method is done at ambient pressure. The solid phase is packed onto a column and the sample is added to bind the protein. It is a very simple method to get purified proteins in a short time.

Limitations of affinity chromatography

Since affinity chromatography solely relies on the interaction of a tag-matrix interaction, it is important to be aware of certain limitations:

Tag accessibility
Interaction between the protein tag and the purification matrix is only possible if the tag is accessible and not buried within the folds of the target protein. Other buffer components such as detergents can also bury the tag sequence and have a negative effect on the tag/matrix interaction. In many cases, moving the tag to the other terminus of the protein - or even to an accessible loop within the protein - can help. If this proves unsuccessful, protein purification via the His-tag (but not via other tags) can also be done under denaturing conditions, thereby exposing the tag to the matrix surface.

Influence of tag on expression
Depending on the protein, tags may interfere with expression rates, folding, and activity. This effect is rather inpredictable unless structural information is already available. Therefore, screening of different tags and tag positions can be useful.

Discrimination between protein conformations / folding variants
Affinity matrices purify all proteins which carry the affinity tag, regardless of correct folding or conformation. To discriminate between different protein conformations, multimers, or to separate correctly folded from inactive protein, protein-specific purification matrices are a useful alternative or additional method.