Affinity Purification of Different Protein Types

Affinity purification is a chromatographic method used to isolate specific proteins and protein complexes from complex biochemical mixtures. The technique is used widely for applications such as purification, pull-down assays, immunoprecipitation (IP), and chromatin immunoprecipitation (ChIP).

The removal of a protein from a mixture, i.e. purification of the protein, is achieved using a protein-specific, reversible reaction between the protein of interest and a linker attached to the beads or resin of a chromatography matrix. As the interaction is specific, the purification is more selective than other chromatographic methods and even small protein volumes can be isolated.

The typical interactions used to purify proteins are ligand-receptor, enzyme-substrate/cofactor/inhibitor and antibody-antigen. In this article, common protein groups will be described that can be isolated using affinity purification techniques.

Purifying Recombinant Proteins

Recombinant proteins tend to be designed to express a tag which makes their purification simpler.

Proteins tagged with histidine can be purified using cobalt or nickel resins whereas glutathione-S-transferase (GST) fusion proteins can be purified using glutathione-based resins like Sepharose® (ab193267).

If you wish to purify a recombinant protein for applications such as immunoprecipitation, you can simply use an antibody raised to the tag. For example, a HA tag with agarose-coupled goat polyclonal (ab1233) or a GFP tag with Sepharose®-coupled rabbit polyclonal.

Purifying Coagulation Factors and Cationic Biomolecules

DNA-binding proteins and coagulation factors, as well as lipoproteins and synthesis factors, can be purified using heparin beads (ab193268). Heparin shows an affinity towards molecules and proteins of a cationic nature meaning heparin resins can also be utilized as cation exchangers of high capacity.

Purifying Antibodies

For antibody purification, IP and ChIP, proteins that are antibody binding, i.e. antigens, can be bound to chromatography beads. Proteins used for this purpose include bacterial proteins (Protein A, Protein A/G, Protein G and Protein L), and a plant-based lectin – jacalin. The specific binding of proteins is discussed in the below table.

When setting up a column for antibody isolation the binding specificity of the protein attached to the resin (antigen) should be matched with the target protein (antibody) intended to capture. To help with this decision process see the information provided in the table below on Protein A, Protein A/G, Protein G, and Protein L), and jacalin:

Immobilized ligand Binding specificity Uses Relevant products**
Protein A Total IgG* IP
IgG purification
Protein A agarose (ab193254)
Protein A agarose (High affinity) (ab193255)
Protein A Sepharose® (ab193256)
Protein A Sepharose® column (ab193257)
Protein G Total IgG* IP
IgG purification
Protein G agarose (high affinity) (ab193258)
Protein G Sepharose®(ab193259)
Protein G Sepharose® column (ab193260)
Protein L Total immunoglobulin, provided immunoglobulin species and subtype contain the appropriate kappa light chains IP
Immunoglobulin purification
Protein L Sepharose®(ab193261)
Protein A/G Binding properties of Protein A and Protein G IP
IgG purification
Protein A/G Sepharose®(ab193262)
Protein A/G Sepharose®column (ab193263)
Protein A/G/L Binding properties of Protein A, Protein G and Protein L IP
Immunoglobulin purification
Protein A/G/L Sepharose®(ab193264)
Protein A/G/L Sepharose®column (ab193265)
Jacalin O-glycoproteins including Human IgA1 and IgD Human IgA purification
Remove of IgA contaminants from IgG
Separation of IgA1 from IgA2
Jacalin Sepharose®(ab193266)

 

*Binding varies by antibody subclass and source species - see additional details here.
** Please review individual datasheets for tested applications and additional specifications.
Sepharose ® is a registered trademark of GE Healthcare.

About Abcam

Abcam is a global life sciences company providing highly validated antibodies and other binders and assays to the research and clinical communities to help advance the understanding of biology and causes of disease.

Abcam’s mission is to serve life scientists to help them achieve their mission faster by listening to their needs, continuously innovating and improving and by giving them the tools, data and experience they want. Abcam’s ambition is to become the most influential life science company for researchers worldwide.


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Last updated: Apr 1, 2019 at 6:12 AM

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