Affinity chromatography: Which tag to use?

Protein purification via affinity chromatography is a powerful technique, and many different affinity tags and resins are available today. This article tries to summarize the main facts to help you choose a tag. However, please keep in mind that the list of affinity tags is constantly growing, and that the information we can provide in this brief overview is far from being complete.

Affinity chromatography: Principles

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 1 for a first overview.

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. Wash:
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
Binding of proteins
Washing of bound proteins
Elution of bound proteins
Fig. 1: Bind wash elute principle exemplified by an antibody-based affinity matrix (e.g. Rho1D4)

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 tags and resins

Table.1: Features of some commercially available affinity tags
Affinity tag His GST Strep® Rho1D4 FLAG®
Affinity tag & size 6-14 histidine residues, 840.8-1,937.9 Da Glutatione S-transferase, 26 kDa protein Strep-tag II: 8 amino acids (WSHPQFEK), 1,058.1 Da Twin-strep tag: 24 amino acids, 2,628.8 Da) 9 amino acids (TETSQVAPA), 902.9 Da 8 amino acids (DYKDDDDK), 1,012.9 Da
Matrix Ni-, Co-, Cu-, or Zn-chelating chemical ligands, e.g. iminodiacetic acid (IDA), nitrilotriacetic acid (NTA) or other ligands with transition-metal binding properties GST Antibody (Glu-Cys-Gly tripeptide) StrepTactin (engineered Streptavidin protein, ca. 15 kDa) Rho1D4 antibody (ca. 150 kDa) DYKDDDDK (FLAG) antibody (ca. 150 kDa)
Elution conditions Imidazole or histidine, or at low pH Reduced glutathione Desthiobiotin or biotin Rho1D4 peptide, low pH, or protease digest DYKDDDDK peptide
Specificity of interaction (KD) 10 µM 1 µM >300 nM (Strep-tag II) ca. 100 nM (Twin-Strep tag) 20 nM (4) 100 nM
Typical protein yield per ml resin up to 80 mg/ml (1) 10-12 mg/ml ca. 3 mg/ml (standard) or up to 9 mg/ml (High Capacity) 3-4 mg/ml 0.6-1 mg/ml
Special features High chemical stability (e.g. DTT, EDTA (1)). Purification can be done under native or denaturing conditions. Enhances solubility for many recombinant proteins. Frequently used for pulldown experiments Can be combined with His-tag purification in a tandem approach Proven track record for membrane proteins (5) Often used for purification from mammalian cells or for immunostaining

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.

Guide to His affinity purification products

Scientists at Cube Biotech purify his-tagged proteins almost every day. New ideas how to optimize the procedure lead to novel products with new exciting features. However, it is difficult to keep track on new developments, and to decide which product to choose for a given application. Therefore, we have built two overview tables for Agaroses and MagBeads that -we hope- are helpful.
Please do not hesitate to contact us directly if you are still unsure, or if you are interested in customized or bulk products.
Table 2: Cube Biotech His Affinity Agarose products.
His Affinity Agarose PureCube 100 INDIGO Ni-Agarose PureCube 100 Ni-NTA Agarose PureCube Ni-NTA Agarose PureCube Ni-NTA Agarose XL PureCube Ni-IDA Agarose
Base material PureCube 100 Agarose PureCube 100 Agarose PureCube Agarose PureCube Agarose XL PureCube Agarose
Bead diameter 50-150 µm 50-150 µm 20-50 µm 300-500 µm 20-50 µm
Ligand Proprietary NTA NTA NTA IDA
Binding capacity up to 80 mg/ml up to 80 mg/ml up to 80 mg/ml 20 mg/ml 50 mg/ml
Purity High High High High Medium
EDTA tolerance 20 mM 1 mM 1 mM 1 mM up to 1 mM
DTT tolerance 20 mM 10 mM 10 mM 10 mM up to 10 mM
pH tolerance 2-13 2-13 2-13 2-13 2-13
Cleaning in place NaOH, GuHCl, Urea, Ethanol NaOH, GuHCl, Urea, Ethanol NaOH, GuHCl, Urea, Ethanol NaOH, GuHCl, Urea, Ethanol NaOH, GuHCl, Urea, Ethanol
Metal stripping No Yes Yes Yes Yes
Flow rate (batch/FPLC) Fast Fast Medium n.a. Medium
Available metal ions Ni Ni, Co Ni, Co, Al Cu, Fe, Zn Ni Ni
Special feature NEW High flow rates, EDTA-stable, compatible with eukaryotic growth media NEW High flow rates, High yields, High purity Standard material, available with various metal ions For special applications The economic alternative
Table 3: Cube Biotech His Affinity MagBead products.
His Affinity Magnetic Beads PureCube INDIGO Ni-MagBeads PureCube Ni-NTA MagBeads PureCube Ni-NTA MagBeads XL PureCube Ni-IDA MagBeads
Base material PureCube MagBeads PureCube MagBeads PureCube MagBeads XL PureCube MagBeads
Bead diameter 25 µm 25 µm 70-120 µm 25 µm
Ligand Proprietary NTA NTA IDA
Binding capacity up to 80 mg/ml up to 80 mg/ml >20 mg/ml >50 mg/ml
Purity High High High Medium
EDTA tolerance 20 mM 1 mM 1 mM up to 1 mM
DTT tolerance 20 mM 10 mM 10 mM up to 10 mM
pH tolerance 2-13 2-13 2-13 2-13
Compatible cleaning agents GuHCl, Urea, Ethanol NaOH, GuHCl, Urea, Ethanol NaOH, GuHCl, Urea, Ethanol NaOH, GuHCl, Urea, Ethanol
Metal stripping with EDTA No Yes Yes Yes
Magnetic separation Fast Fast Medium Fast
Available metal ions Ni Ni, Co, Al Cu, Fe, Zn Ni Ni
Special feature EDTA-stable, compatible with eukaryotic growth media. High yield & purity, Broad ion variety For special applications Larger beads on request The economic alternative

NTA or IDA resin for His tag purification

Choosing the right ligand for the hig tag purification products, can be confusing as NTA and IDA seem to be nearly identical at the first look. For this reason we wrote THIS GUIDE about the differences between NTA and IDA.
 NTA and IDA differenceFig 2: IDA and NTA both form a chelator complex with a metal ion that in turn can interact with two histidines of a poly-his tag on a protein of interest.

Further reading

1. IDA vs NTA: A tale of two ligands, Cube Biotech 2013
2. Angelo DePalma, PhD: Poly-His Tags Improve Protein Purification. The most popular protein tagging method has many advantages and few caveats. Genetic Engineering and Biotechnology News May 8, 2014
3. Angelo DePalma, PhD: Getting a Sure Hold on Protein Purification by Attaching Convenient Handles. Genetic Engineering and Biotechnology News Mar 1, 2015, Vol. 35, No. 5
4. Locatelli-Hoops, S.C. 2013. Expression, surface immobilization, and characterization of functional recombinant cannabinoid receptor CB2. Biochim. Biophys. Acta 1834 (10):2045-56.
5. PureCube Rho1D4 Agarose, Cube Biotech 2013
Trademarks and disclaimers: StrepTactin® and Strep-tag®(IBA), FLAG® (Sigma-Aldrich Co. LLC)