Protein Affinity Chromatography
Affinity chromatography is the preferred method of bioselective adsorption and subsequent recovery of a compound from an immobilized ligand. Each is designed for highly specific and efficient purification of proteins and related compounds. We offer appropriately selective ligands on beaded and porous matrices for binding target compounds, which are then recovered under mild conditions. Your choice for optimal downstream utility.
Affinity Class | Potential Application |
---|---|
Activated / Functionalized | Functional spacer; support matrix; eliminates handling of toxic reagents |
Amino Acid | Serum proteins; proteins; peptides; enzymes; rRNA; dsDNA |
Avidin Biotin | Purification of biotin/avidin & derivatives; biotinylated substances. Biotin derivatives dissociate under nondenaturing conditions. |
Carbohydrate Binding | Solube glycoproteins ; other carbohydrate-containing substances |
Carbohydrate | Glycoproteins; lectins; other carbohydrate metabolite proteins. Proper selection can ensure one-step purification. |
Dye Ligand | Nonspecific interaction. Mimic biological substrates (substrates, cofactors, effetors); proteins. Optimize purification protocol with different dyes |
Glutathione | Purification of glutathione enzymes and GST tagged recombinant proteins |
Heparin | General affinity ligand, useful for plasma coagulation proteins, nucleic acid enzymes, lipases, etc. |
Hydrophobic Interactions | Couple ligands containing free carboxyl groups; proteins |
IMAC | Immobilized Metal Affinity Chromatography. Uses interactions between protein and chelated metal to separate. |
Immunoaffinity | Quantitative determinination of antigens, high specificity |
Nucleotide / Coenzyme | Dehydrogenases; kinases; transaminases. Reliable adsorbents. |
Nucleic Acid | mRNA; DNA; rRNA; other nucleic acids and oligonucleotides |
Protein A / Protein G | Purification of immunoglobulins |
Speciality | Purification of specific classes or types of proteins, coenzymes, or physiological partners |
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.
Activation | Linkage to Resin | Available Reactive Group | Specificity of Group | Reaction Conditions | Bond Type; Stability of Attachment | Intrinsic Spacer |
---|---|---|---|---|---|---|
Carbonyldimidazole | Carbamate | Imidazole Carbamate | Amine | pH 8-10 | Carbamate; good stability below pH 10 | 1 atom neutral |
Cyanogen Bromide | Final isourea | Cyanate ester | Amine | pH 8-9.5 | Isourea; moderately stable | 1 atom cationic |
Epichlorohydrin | Ether | Epoxy | SH>NH2>OH | pH 7-8 SH | Thioether sec amine ether; all very stable | 3 atoms neutral |
Epoxy (bis) | Ether | Epoxy | SH>NH2>OH | pH 7-8 SH pH 9-11 NH2 pH >11 OH | Thioether sec amine ether; all very stable | 12 atoms neutral |
N-Hydroxy-succinimidyl Chloroformate | Final carbonate | Succinimidyl Carbonate | Amine | pH 8-9.5 | Carbamate; good stability below pH 10 | 1 atom neutral |
p-Nitrophenyl Chloroformate | Final carbonate | Nitrophenyl Carbonate | Amine | pH 8.5-10 | Carbomate; good stability below pH 10 | 1 atom neutral |
Tresyl Chloride | Alkylamine thioether | Tresyl sulfonate | Amine, thiol | pH 7-9.5 | Alkylamine thioether; both very stable | 0 atoms direct linkage |
Vinyl Sulfone | Sulfonyl thioether | Vinyl sulfone | SH>NH2>OH | pH 6-8 SH pH 8-10 NH2 pH >10 OH | ThiotherSec amine ether; Good stability below pH 9 | 5 atoms neutral, some non-specific effects |
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:
Active Group | Specificity | Coupling Conditions | Bond to ligand; stability |
---|---|---|---|
N-Hydroxysuccinimide Ester; active ester | Amine | pH 6.0-8.0 | Amine; good stability |
Disulfide; reactivity based on leaving group | Sulfhydryl | pH 6.0-8.0 | Covalent disulfide; good stability under nonreducing conditions |
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
Sugar Specificity | Lectin |
---|---|
β-D-gal(1-3)-D-galNAc | Arahis hypogaea (Peanut) |
Methyl-α-D-gal | Artocarpus integrifolia (Jacalin) |
α-D-man, α-D-glc | Concanavalin A Type VI (Con A) |
α-D-man, α-D-glc | Succinyl Concanvalin A |
Nonreducing end of terminal α-D-mannosyl residue of glycoconjugates | Galanthus nivalis (Snowdrop) |
D-galNAc | Glycine max (Soybean) |
D-galNAc | Helix pomatia (Roman or edible snail) |
α-D-man | Lens culinaris (Lentil) |
Oligosaccharide | Phaseolus vulgaris PHA-E (Red kidney bean) |
(D-glcNAc)2, NeuNAc | Triticum vulgaris (Wheat germ) |
α-L-fuc | Ulex europaeus UEA-1 (gorse of Furze) |
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.
Metal | Application |
---|---|
Cobalt | Purification of HIS-tagged proteins |
Nickel | Purification of HIS-tagged proteins |
Copper | Usually for purification of HIS-tagged proteins and histidine containing peptides |
Zinc | Usually for the purification of HIS-tagged proteins |
Iron | Purification of Phosphopeptides |
Gallium | Purification of Phosphopeptides |
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
Resin Type | For the purification of (Applications), [Reference] |
---|---|
Albumin agarose | Steroids, amino acids, bilirubin & heme peptides (For general hydrophobic adsorption & chiral resolution) 1,2 |
p-Aminobenzamide agarose | Serine proteases, urokinases, plasminogen activators and protease removal 3,4 |
m-Aminophenylboronic acid agarose | Glycoproteins (This matrix offers high capacity) 5 |
N(p-Aminophenyl)oxamic acid agarose | Neuraminidase & influenza virus 6 |
Antithrombin III agarose | Heparin & thrombin 7,8 |
Aprotinin agarose | Serine proteases, urokinases & protease removal 9 |
Cholesterol hemisuccinate agarose | HDL, LDL & cholesterol oxidase 10 |
α-Cobrotoxin agarose | Nicotinic acetylcholine (snake toxin) receptors 12 |
Fetuin agarose | Lectins, glycosidases & carbohydrate binding proteins 12,13 |
Folic acid agarose | Folate binding proteins 14 |
Forskolin agarose | Adenylate cyclase 15 |
Gelatin agarose | Fibronectin 16,17 |
Hemoglobin agarose | Hemoglobin based O2 carriers 18 |
L-Histidyldiazobenzyl phophonic acid agarose | Phosphatases 19 |
Histone agarose | Protein kinases 20 |
Methotrexate agarose | Dihydrofolate reductase 21 |
Pepstatin A agarose | Cathepsin D & Rennin 22 |
Phosphodiesterase 3’-5’ cyclic nucleotide activator agarose | Calmodulin binding proteins 23,24 |
O-Phospho-L-Tyrosine agarose | Phosphatases 19 |
Poly-L-Lysine agarose | DNA, RNA & T4 phage 25 |
Polymyxin B agarose | Lipopolysaccharides (endotoxins) 26,27 |
Tartaric acid agarose | Prostatic acid phosphatases 30,31 |
Trypsin inhibitor agarose | Proteases or protease removal 32,33 |
References
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