All tags proteomics-tools Overview of affinity purification

Overview of affinity purification

Affinity purification is used to separate a protein or protein complex of interest from a biochemical mixture in a wide range of applications, including protein purification, immunoprecipitation (IP), chromatin immunoprecipitation (ChIP) and pull-down assays. 

Purification is achieved through a specific, reversible reaction between your protein of interest and a ligand that is covalently attached through a linker arm to a chromatography matrix (beads or resins), such as agarose or Sepharose® beads. The specificity of this interaction enables more selective separation than other chromatography methods and allows you to purify relatively small amounts of your protein(s) of interest. Typical interactions that are exploited in affinity purification include antibody-antigen, enzyme-substrate/inhibitor/cofactor and ligand-receptor. We review the most common groups of proteins that are isolated using affinity purification beads (also termed affinity purification resins) below. 

Recombinant fusion proteins

Recombinant proteins are often expressed with a tag to facilitate their purification. Glutathione-S-Transferase (GST) fusion proteins are purified using glutathione resins, such as glutathione Sepharose® (ab193267) Histidine-tagged proteins are typically purified using nickel or cobalt resins. If you wish to purify a recombinant protein for applications such as immunoprecipitation, you can simply use an antibody raised to the tag, e.g., Sepharose®-coupled rabbit polyclonal to GFP (ab69314) or  agarose-coupled goat polyclonal to HA tag (ab1233). Find out more about our complete range of anti-tag antibodies here.

Coagulation factors and cationic biomolecules

Heparin beads (ab193268) are used to purify a range of proteins, including DNA-binding proteins, coagulation factors, lipoproteins and protein synthesis factors. As heparin binds to proteins or biomolecules with an overall positive charge, you can also use heparin resins as high capacity cation exchangers.  

Heparin beads ( ab193268) are used to purify a range of proteins, including DNA-binding proteins, coagulation factors, lipoproteins and protein synthesis factors. As heparin binds to proteins or biomolecules with an overall positive charge, you can also use heparin resins as high capacity cation exchangers.  

Antibodies

Several antibody-binding proteins are coupled to beads for use in antibody purification, IP and ChIP. These include Protein A, Protein G, Protein A/G and Protein L, which are proteins of bacterial origin, and jacalin, a plant-based lectin. The binding specificities of these proteins are summarized in the table below.

When designing an experiment to isolate antibodies from a mixture, you should match the binding specificity of the immobilized protein to the species and antibody subtype of the antibody you wish to capture. To help you decide which protein resins to use, we have compiled detailed information about the species and antibody subtype binding properties for Protein A, Protein G, Protein A/G, Protein L and  jacalin here

​Prefer to use a kit with an optimized protocol for antibody purification? View our antibody purification kits here.

Immobilized ligandBinding specificityUsesRelevant products**
Protein ATotal IgG*IP
IgG purification
Protein A agarose (ab193254)

Protein A agarose (High affinity) (ab193255)

Protein A Sepharose®
(ab193256)


Protein A Sepharose®
column (ab193257)
Protein GTotal IgG*IP  
IgG purification

Protein G agarose (high affinity) (ab193258)

Protein G Sepharose® (ab193259)

Protein G Sepharose®​ column (ab193260)

Protein LTotal immunoglobulin, provided immunoglobulin species and subtype contain the appropriate kappa light chainsIP
Immunoglobulin purification

Protein L Sepharose® (ab193261)


Protein A/GBinding properties of Protein A and Protein GIP
IgG purification

Protein A/G Sepharose® (ab193262)

Protein A/G Sepharose® column (ab193263)

Protein A/G/LBinding properties of Protein A, Protein G and Protein LIP
Immunoglobulin purification

Protein A/G/L Sepharose® (ab193264)

Protein A/G/L Sepharose® column (ab193265)

JacalinO-glycoproteins including Human IgA1 and IgDHuman 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. 





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