Protein Affinity Chromatography

Activated / Functionalized Matrices

The availability of pre-activated supports allows the user to quickly and easily custom synthesize affinity resins without having to handle highly reactive reagents. We offer a variety of activated matrices ready for direct coupling to a diversity of ligands. The ligand, generally, must possess a free primary amine, sulfhydryl, or hydroxyl group for direct attachment. Ligand characteristics will be a major determinant in choosing an appropriate matrix.

Historically, cyanogen bromide activated matrices have been very popular. Their ability to efficiently couple amine functionalities under mild conditions has, for many, offset the problems of ligand leaching and ionic properties associated with this chemistry. We offer many alternative activated resins with coupling chemistries suitable for stable attachment to most types of potential ligands. Different activating chemistries will impart unique characteristics to the reactivity and functionality of the affinity matrix. The major properties for each direct activation type are described in the accompanying table.

Some activated matrices are available which contain an additional spacer due to prior derivatization. These resins may permit the use of milder coupling conditions or a more specific attachment of a ligand. Examples of these are as follows:

We offer a variety of matrices with incorporated terminal groups suitable for custom derivatization or coupling. Typically these groups will be a carboxyl or amine function, which may be coupled by an amide bond to a ligand, and require an additional reagent to accomplish condensation. Another group commonly utilized is hydrazide, which may couple to aldehydes by hydrazone formation and typically does not require additional reagent.

Amino Acid Resins

We offer a variety of amino acids coupled through the amino group to cyanogens bromide activated 4% beaded agarose. These resins may be used directly for adsorpotion of compounds having an affinity for the particular amino acid immobilized, or they can serve as functionalised linkers and allow covalent attachment through their free carboxyl terminus to a ligand containing a free primary amine.

Avidin / Biotin Matrices

The protein Avidin has an extremely high affinity for the cofactor Biotin (Kdiss 10-15M). This characteristic provides a unique and powerful tool, which can be used for separation and purification. Many compounds are easily biotinylated with retention of biological activity, allowing for a broad range of separation applications. We offer a wide variety of biotinylated products, biotinylation reagents and avidin derivates.

One disadvantage of the strong interaction between avidin and biotin is the harsh denaturing conditions required to accomplish dissociation. Harsh conditions may be avoided by using a biotin derivative such as 2-iminobiotin, which dissociates from avidin under milder conditions. Another alternative, is the use of monomeric avidin, which has a much lower affinity for biotin than the natural tetramer. This allows for dissociation of the complex by competitive displacement.

Carbohydrate Binding Matrices

Lectins are the proteins that have the ability to bind to sugar moieties and agglutinate cells. Once immobilized, lectins can be utilized for the purification of selected glycoconjugates, which may generally be recovered by competitive displacement using an inhibitory simple sugar. Lectin resins have been used to:

• Purify polysaccharides
• Fractionate cell parts
• Purify glycoproteins and other glycoconjugate molecules
• Remove carbohydrate containing impurities from a protein solution

Carbohydrate Matrices

Immobilized sugars and sugar derivatives offer an effective means of purification for a variety of proteins, which recognize and bind carbohydrate moieties. Among the most widely used applications for these matrices are the purification of lectins, glycosidases, and carbohydrate directed antibodies. Lectin isolation is of particular interest in that they are highly useful for studies of cell membrane structure and function, determination of blood groups, cell fractionation, and stimulation of lymphocyte mitosis.

Dye Ligand Resins

Dye ligand chromatography is affinity chromatography that utilizes covalently bond textile dye (reactive dyes) to purify proteins. These dyes resemble natural substrates which proteins have affinities for, thus dye ligand chromatography is sometimes referred to as pseudo-affinity chromatography. Immobilized dyes are not specific adsorbents, but rather have a broad binding range for a variety of proteins. After careful investigation of the proper binding and elution conditions, a high degree of purification of a target protein can be attained even in the presence of a mixture of other proteins.

Glutathione Resins

Glutathione is a tri-peptide, which consists of the amino acids Glutamic acid, Cysteine and Glycine. Glutathione and its derivatives are very useful as ligands in affinity chromatography for the isolation of Glutathione requiring enzymes. These include detoxification enzymes such as: Glutathione Transferase, Glutathione Peroxidase and Glyoxalase I. In recent years, Glutathione agarose resins have become useful for the isolation of fusion proteins due to the presence of Glutathione transferase (GST) as part of the fusion protein. By fusing GST to recombinant proteins, Glutathione resins can separate a selected target protein from other components.

Heparin Resins

Heparin is a highly sulphated glycosaminoglycan, which has widespread use as a general affinity ligand. Its high degree of sulfation imparts a strong acidic nature to the molecule, therefore it may bind to many substances by ionic interaction. In addition, heparin contains unique carbohydrate sequences, which act as specific binding sites for some proteins. Immobilized heparin has been used to purify plasma coagulation proteins, nucleic acid enzymes, lipases, and other proteins.

Hydrophobic Interaction Resins

Hydrophobic chromatography is a versatile technique used for the separation and purification of proteins based on their hydrophobicity. Columns are usually run under conditions that favor hydrophobic interaction, such as high ionic strength. This makes hydrophobic chromatography an ideal tool to use following salt precipitation. Our hydrophobic gels are available in three functional variations:

• Pure hydrophobic resins—these gels contain ligands attached through a stable ether linkage and they contain no charged regions
• Mixed property resins—these gels contain ligands attached through an amine group and in some cases they will carry a charge
• Aminoalkyl resins—these gels provide a hydrophobic spacer to which other functional groups may be coupled

IMAC Matrices

Immobilized Metal Affinity Chromatography (IMAC) is the process of protein separation based on the differential interaction of various proteins with different chelated / insolubilized metals. This interaction depends on:

• The relative amount and location of certain amino acids within the protein (namely histidine, cysteine, tyrosine, and typtophan)
• The type of metal utilized

Generally, different transition metals may be chelated to and exchanged within the same resin, however, some chelating groups may demonstrate preferential chelation of certain metals. Commonly utilized metals are: Copper, Zinc, Iron, and Nickel.

Immunoaffinity Matrices

Immobilized antibodies are used as analytical tools for the quantitative determination of antigens, including immunoglobulins and haptens. Immunoprecipitation followed by SDS-PAGE is a technique typically used to determine the quantity and presence of an antigen in a complex protein mixture such as a cell lysate. Cells may be separated according to surface antigens using various immobilized immunochemicals. Antibody-agarose products are also used in a variety of application including immunoadsorption, affinity chromatography and as solid-phase secondary antibodies. Due to this diversity, we offer a wide selection of antibodies and whole sera immobilized onto 4% crosslinked agarose.

Nucleotide / Coenzyme Resins

Cofactors, coenzymes, and substrates bound to matrices play a major role in protein purification. Since nearly one third of known enzymes require a nucleotide coenzyme, nucleotide bound resins are useful for the purification of many proteins. Protein binding to a nucleotide coenzyme is dependent on spacer arm length and the position of ligand attachment. We offer a wide variety of nucleotide resins with different attachment positions and spacer arms to provide the chemist greater flexibility in affinity fractionations.

Protein A / Protein G Matrices

Protein A and Protein G are bacterial proteins, which demonstrate specific binding to the Fc (non-antigen binding) portion of many classes of immunoglobulins. Protein A and G affinity matrices have been used primarily for:

• Affinity purification of immunoglobulin, primarily IgGs
• Separation of Fc from Fab fragments

Protein A resins have historically been popular for most potential applications, however it has been demonstrated that Protein G resin can enhance and broaden the scope of application. The binding characteristics of the two proteins for various types of immunoglobulins vary and may be used to good advantage. Some of the major differences in binding are:

• Protein A
  • Broad species reactivity; binds well to IgG from human, rabbit, cow and guinea pig
  • Weak binding to monoclonal antibodies
    
• Protein G
  • Broader species reactivity; much stronger binding to IgG from mouse, rat and goat
  • Stronger binding to monoclonal antibodies

Specialty Resins

The following is a sample list of products containing a variety of specialty affinity matrices. Each resin is listed with possible applications and references.