Affinity chromatography products

Affinity chromatography selection guide

To find the media or prepacked columns that suit your needs, choose the option below that best describes your application.

What type of protein will you purify?

We offer products for affinity purification of:

  • Tagged proteins: histidine-tagged (his-tagged), glutathione S-transferase (GST)-tagged, maltose binding protein (MBP)-tagged, Strep II-tag tagged.
  • Antibodies and antibody fragments.
  • Proteins that have affinity for specific groups of molecules. We provide options for affinity purification (enrichment) or removal of proteins containing biotin or carbohydrate groups. Media for affinity purification of other proteins are also available. Please see our selection tool for options.
  • Proteins with affinity for a ligand that can be coupled to GE pre-activated media. Options for coupling of a ligand via an amino, thiol, carboxyl, or hydroxyl group are available.

Purification of His-tagged proteins

We offer a variety of products containing IMAC media precharged with nickel or cobalt ions. These products include prepacked columns for use with chromatography systems, prepacked formats for manual purification, and loose media (resins). Options containing uncharged IMAC media are also available. We recommend products containing the following media types based on your purification priorities:

  • High purity: TALON Superflow, which is precharged with cobalt ions
  • Avoiding nickel leakage: Ni Sepharose excel, which is precharged with nickel ions
  • High binding capacity: Ni Sepharose High Performance, which is precharged with nickel ions
  • Ability to scale up purification: Ni Sepharose 6 Fast Flow, which is precharged with nickel ions
  • Flexibility in metal ions used: Uncharged IMAC media

The choice of metal ion affects binding specificity and strength. The requirements for this purification step will help with this choice. Nickel (Ni2+) is the most popular metal ion for histidine-tagged (his-tagged) protein purification. But nickel tends to bind nonspecifically to host proteins containing histidine clusters. Cobalt (Co2+) interacts more specifically with his-tags, often giving a more pure protein. GE offers many products containing media precharged with nickel or cobalt ions. Uncharged IMAC media are flexible options for preparing media charged with metal ions other than nickel or cobalt, such as zinc or copper.

Before applying a crude sample to an IMAC column, the sample is typically clarified using centrifugation and filtration. An alternative is to apply an unclarified, crude sample to an appropriate column directly after thorough cell disruption. This option can save time and help to minimize protein degradation. GE provides a wide range of products that have been designed to purify histidine-tagged proteins from unclarified samples.

Mammalian (e.g., CHO cells) and insect cell culture media contain substances that could reduce the yield of purified histidine-tagged (his-tagged) proteins. These components lower the binding to the IMAC medium by stripping the chelated metal ion. To improve protein yield, the sample can be pretreated to remove the interfering substances. Another option is to use a medium (e.g., Ni Sepharose excel) that binds to his-tagged proteins in the presence of metal stripping agents.

Purification of antibodies and antibody fragments

Our product offering includes a range of agarose-based media with protein A, protein G, or protein L as ligand. We provide a variety of protein A- and G-based products for purifying monoclonal and polyclonal antibodies from cell culture supernatants and serum. Capto L is our medium for purifying antibody fragments that contain a kappa light chain. These fragments include single-chain fragment variable (scFv), Fab, and domain antibodies (Dab). Most of our products are available as prepacked columns for use with chromatography systems, prepacked formats for manual purification, and loose media (resins). We also offer prepacked columns for IgM and IgY purification, which are based on thiophilic adsorption.

Please use our online selection tool to find a product that meets your needs.

Both protein A and protein G have high affinity and specificity for the Fc region of antibodies. However, they differ in their ability to bind antibodies of different species and subclasses (see table). We recommend protein A for purifying human antibodies. Protein G binds a broader range of IgG from eukaryotic species and binds more classes of IgG than protein A. Therefore, we recommend our protein G-based media to purify antibodies from other species, including rat. If you are not sure whether your antibody will bind to protein A or protein G, we suggest trying rProtein A/Protein G GraviTrap, which contains gravity flow columns packed with a combination of both media.

The table below shows the relative binding strengths of antibodies from various species to protein G and protein A.

Relative binding strengths of antibodies from various species to protein G and protein A as measured in a competitive ELISA test. The amount of IgG required to give a 50% inhibition of binding of rabbit IgG conjugated with alkaline phosphatase was determined.

Species Subclass Protein G binding Protein A binding

* Purified using HiTrap IgM Purification HP columns

Purified using HiTrap IgY Purification HP columns

++++ = strong binding

++ = medium binding

— = weak or no binding

Human IgA variable
IgD
IgE
IgG1 ++++ ++++
IgG2 ++++ ++++
IgG3 ++++
IgM* variable
IgG4 ++++ ++++
Avian egg yolk IgY
Cow ++++ ++
Dog + ++
Goat ++
Guinea pig IgG1 ++ ++++
Hamster ++ +
Horse ++++ ++
Koala +
Llama +
Monkey (rhesus) ++++ ++++
Mouse IgG1 ++++ +
IgG2a ++++ ++++
IgG2 +++ +++
IgG3 +++ ++
IgM* variable
Pig +++ +++
Rabbit +++ ++++
Rat IgG1 +
IgG2a ++++
IgG2b ++
IgG3 ++ +
Sheep ++ +/—

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