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Affinity chromatography
Affinity chromatography
Affinity chromatography
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Affinity chromatography is a method of separating a biomolecule from a mixture, based on a highly specific macromolecular binding interaction between the biomolecule and another substance. The specific type of binding interaction depends on the biomolecule of interest; antigen and antibody, enzyme and substrate, receptor and ligand, or protein and nucleic acid[1] binding interactions are frequently exploited for isolation of various biomolecules. Affinity chromatography is useful for its high selectivity and resolution of separation,[2][3] compared to other chromatographic methods.

Principle

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Affinity chromatography has the advantage of specific binding interactions between the analyte of interest (normally dissolved in the mobile phase), and a binding partner or ligand (immobilized on the stationary phase). In a typical affinity chromatography experiment, the ligand is attached to a solid, insoluble matrix—usually a polymer such as agarose or polyacrylamide—chemically modified to introduce reactive functional groups with which the ligand can react, forming stable covalent bonds.[4] The stationary phase is first loaded into a column to which the mobile phase is introduced. Molecules that bind to the ligand will remain associated with the stationary phase. A wash buffer is then applied to remove non-target biomolecules by disrupting their weaker interactions with the stationary phase, while the biomolecules of interest will remain bound. Target biomolecules may then be removed by applying a so-called elution buffer, which disrupts interactions between the bound target biomolecules and the ligand. The target molecule is thus recovered in the eluting solution.[5][page needed]

Affinity chromatography does not require the molecular weight, charge, hydrophobicity, or other physical properties of the analyte of interest to be known, although knowledge of its binding properties is useful in the design of a separation protocol.[5] Types of binding interactions commonly exploited in affinity chromatography procedures are summarized in the table below.

Typical biological interactions used in affinity chromatography[6]
Sr. no Types of ligand Target molecule
1 Substrate analogue Enzymes
2 Antibody Antigen
3 Lectin Polysaccharide
4 Nucleic acid Complementary base sequence
5 Hormone Receptor
6 Avidin Biotin/Biotin-conjugated molecule
7 Calmodulin Calmodulin binding partner
8 Glutathione GST fusion protein
9 Protein A or Protein G Immunoglobulins
10 Nickel-NTA polyhistidine fusion protein

Batch and column setups

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Principle of affinity column chromatography
Batch chromatography

Binding to the solid phase may be achieved by column chromatography whereby the solid medium is packed onto a column, the initial mixture run through the column to allow settling, a wash buffer run through the column and the elution buffer subsequently applied to the column and collected. These steps are usually done at ambient pressure. Alternatively, binding may be achieved using a batch treatment, for example, by adding the initial mixture to the solid phase in a vessel, mixing, separating the solid phase, removing the liquid phase, washing, re-centrifuging, adding the elution buffer, re-centrifuging and removing the elute.[citation needed]

Sometimes a hybrid method is employed such that the binding is done by the batch method, but the solid phase with the target molecule bound is packed onto a column and washing and elution are done on the column.[citation needed]

The ligands used in affinity chromatography are obtained from both organic and inorganic sources. Examples of biological sources are serum proteins, lectins and antibodies. Inorganic sources are moronic acid, metal chelates and triazine dyes.[7]

A third method, expanded bed absorption, which combines the advantages of the two methods mentioned above, has also been developed. The solid phase particles are placed in a column where liquid phase is pumped in from the bottom and exits at the top. The gravity of the particles ensure that the solid phase does not exit the column with the liquid phase.[citation needed]

Affinity columns can be eluted by changing salt concentrations, pH, pI, charge and ionic strength directly or through a gradient to resolve the particles of interest.[citation needed]

More recently, setups employing more than one column in series have been developed. The advantage compared to single column setups is that the resin material can be fully loaded since non-binding product is directly passed on to a consecutive column with fresh column material. These chromatographic processes are known as periodic counter-current chromatography (PCC). The resin costs per amount of produced product can thus be drastically reduced. Since one column can always be eluted and regenerated while the other column is loaded, already two columns are sufficient to make full use of the advantages.[8] Additional columns can give additional flexibility for elution and regeneration times, at the cost of additional equipment and resin costs.[citation needed]

Specific uses

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Affinity chromatography can be used in a number of applications, including nucleic acid purification, protein purification[9] from cell free extracts, and purification from blood.

By using affinity chromatography, one can separate proteins that bind to a certain fragment from proteins that do not bind that specific fragment.[10] Because this technique of purification relies on the biological properties of the protein needed, it is a useful technique and proteins can be purified many folds in one step.[11][page needed]

Various affinity media

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Many different affinity media exist for a variety of possible uses.[12][9][13] Briefly, they are (generalized) activated/functionalized that work as a functional spacer, support matrix, and eliminates handling of toxic reagents.[citation needed]

Amino acid media is used with a variety of serum proteins, proteins, peptides, and enzymes, as well as rRNA and dsDNA. Avidin biotin media is used in the purification process of biotin/avidin and their derivatives.[citation needed]

Carbohydrate bonding is most often used with glycoproteins or any other carbohydrate-containing substance; carbohydrate is used with lectins, glycoproteins, or any other carbohydrate metabolite protein. Dye ligand media is nonspecific but mimics biological substrates and proteins. Glutathione is useful for separation of GST tagged recombinant proteins. Heparin is a generalized affinity ligand, and it is most useful for separation of plasma coagulation proteins, along with nucleic acid enzymes and lipases[citation needed]

Hydrophobic interaction media are most commonly used to target free carboxyl groups and proteins.[citation needed]

Immunoaffinity media (detailed below) utilizes antigens' and antibodies' high specificity to separate; immobilized metal affinity chromatography is detailed further below and uses interactions between metal ions and proteins (usually specially tagged) to separate; nucleotide/coenzyme that works to separate dehydrogenases, kinases, and transaminases.[citation needed]

Nucleic acids function to trap mRNA, DNA, rRNA, and other nucleic acids/oligonucleotides. Protein A/G method is used to purify immunoglobulins.[citation needed]

Speciality media are designed for a specific class or type of protein/co enzyme; this type of media will only work to separate a specific protein or coenzyme.[citation needed]

Immunoaffinity

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Another use for the procedure is the affinity purification of antibodies from blood serum. If the serum is known to contain antibodies against a specific antigen (for example if the serum comes from an organism immunized against the antigen concerned) then it can be used for the affinity purification of that antigen. This is also known as Immunoaffinity Chromatography. For example, if an organism is immunised against a GST-fusion protein it will produce antibodies against the fusion-protein, and possibly antibodies against the GST tag as well. The protein can then be covalently coupled to a solid support such as agarose and used as an affinity ligand in purifications of antibody from immune serum.[citation needed]

For thoroughness, the GST protein and the GST-fusion protein can each be coupled separately. The serum is initially allowed to bind to the GST affinity matrix. This will remove antibodies against the GST part of the fusion protein. The serum is then separated from the solid support and allowed to bind to the GST-fusion protein matrix. This allows any antibodies that recognize the antigen to be captured on the solid support. Elution of the antibodies of interest is most often achieved using a low pH buffer such as glycine pH 2.8. The eluate is collected into a neutral tris or phosphate buffer, to neutralize the low pH elution buffer and halt any degradation of the antibody's activity. This is a nice example as affinity purification is used to purify the initial GST-fusion protein, to remove the undesirable anti-GST antibodies from the serum and to purify the target antibody.[citation needed]

Monoclonal antibodies can also be selected to bind proteins with great specificity, where protein is released under fairly gentle conditions. This can become of use for further research in the future.[14]

A simplified strategy is often employed to purify antibodies generated against peptide antigens. When the peptide antigens are produced synthetically, a terminal cysteine residue is added at either the N- or C-terminus of the peptide. This cysteine residue contains a sulfhydryl functional group which allows the peptide to be easily conjugated to a carrier protein (e.g. Keyhole limpet hemocyanin (KLH)). The same cysteine-containing peptide is also immobilized onto an agarose resin through the cysteine residue and is then used to purify the antibody.[citation needed]

Most monoclonal antibodies have been purified using affinity chromatography based on immunoglobulin-specific Protein A or Protein G, derived from bacteria.[15]

Immunoaffinity chromatography with monoclonal antibodies immobilized on monolithic column has been successfully used to capture extracellular vesicles (e.g., exosomes and exomeres) from human blood plasma by targeting tetraspanins and integrins found on the surface of the EVs.[16][17]

Immunoaffinity chromatography is also the basis for immunochromatographic test (ICT) strips, which provide a rapid means of diagnosis in patient care. Using ICT, a technician can make a determination at a patient's bedside, without the need for a laboratory.[18] ICT detection is highly specific to the microbe causing an infection.[19]

Immobilized metal ion affinity chromatography

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Immobilized metal ion affinity chromatography (IMAC) is based on the specific coordinate covalent bond of amino acids, particularly histidine, to metals. This technique works by allowing proteins with an affinity for metal ions to be retained in a column containing immobilized metal ions, such as cobalt, nickel, or copper for the purification of histidine-containing proteins or peptides, iron, zinc or gallium for the purification of phosphorylated proteins or peptides. Many naturally occurring proteins do not have an affinity for metal ions, therefore recombinant DNA technology can be used to introduce such a protein tag into the relevant gene. Methods used to elute the protein of interest include changing the pH, or adding a competitive molecule, such as imidazole.[20][21]

A chromatography column containing nickel-agarose beads used for purification of proteins with histidine tags
See also: Polyhistidine-tag

Recombinant proteins

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Possibly the most common use of affinity chromatography is for the purification of recombinant proteins. Proteins with a known affinity are protein tagged in order to aid their purification. The protein may have been genetically modified so as to allow it to be selected for affinity binding; this is known as a fusion protein. Protein tags include hexahistidine (His), glutathione-S-transferase (GST), maltose binding protein (MBP), and the Colicin E7 variant CL7 tag. Histidine tags have an affinity for nickel, cobalt, zinc, copper and iron ions which have been immobilized by forming coordinate covalent bonds with a chelator incorporated in the stationary phase. For elution, an excess amount of a compound able to act as a metal ion ligand, such as imidazole, is used. GST has an affinity for glutathione which is commercially available immobilized as glutathione agarose. During elution, excess glutathione is used to displace the tagged protein. CL7 has an affinity and specificity for Immunity Protein 7 (Im7) which is commercially available immobilized as Im7 agarose resin. For elution, an active and site-specific protease is applied to the Im7 resin to release the tag-free protein.[22]

Lectins

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Lectin affinity chromatography is a form of affinity chromatography where lectins are used to separate components within the sample. Lectins, such as concanavalin A are proteins which can bind specific alpha-D-mannose and alpha-D-glucose carbohydrate molecules. Some common carbohydrate molecules that is used in lectin affinity chromatography are Con A-Sepharose and WGA-agarose.[23] Another example of a lectin is wheat germ agglutinin which binds D-N-acetyl-glucosamine.[24] The most common application is to separate glycoproteins from non-glycosylated proteins, or one glycoform from another glycoform.[25] Although there are various ways to perform lectin affinity chromatography, the goal is extract a sugar ligand of the desired protein.[23]

Specialty

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Another use for affinity chromatography is the purification of specific proteins using a gel matrix that is unique to a specific protein. For example, the purification of E. coli β-galactosidase is accomplished by affinity chromatography using p-aminobenyl-1-thio-β-D-galactopyranosyl agarose as the affinity matrix. p-aminobenyl-1-thio-β-D-galactopyranosyl agarose is used as the affinity matrix because it contains a galactopyranosyl group, which serves as a good substrate analog for E. coli β-Galactosidase. This property allows the enzyme to bind to the stationary phase of the affinity matrix and β-Galactosidase is eluted by adding increasing concentrations of salt to the column.[26]

Alkaline phosphatase

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Alkaline phosphatase from E. coli can be purified using a DEAE-Cellulose matrix. A. phosphatase has a slight negative charge, allowing it to weakly bind to the positively charged amine groups in the matrix. The enzyme can then be eluted out by adding buffer with higher salt concentrations.[27]

Boronate affinity chromatography

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Boronate affinity chromatography consists of using boronic acid or boronates to elute and quantify amounts of glycoproteins. Clinical adaptations have applied this type of chromatography for use in determining long term assessment of diabetic patients through analysis of their glycated hemoglobin.[24]

Serum albumin purification

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Affinity purification of albumin and macroglobulin contamination is helpful in removing excess albumin and α2-macroglobulin contamination, when performing mass spectrometry. In affinity purification of serum albumin, the stationary used for collecting or attracting serum proteins can be Cibacron Blue-Sepharose. Then the serum proteins can be eluted from the adsorbent with a buffer containing thiocyanate (SCN−).[28]

Weak affinity chromatography

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Weak affinity chromatography[29] (WAC) is an affinity chromatography technique for affinity screening in drug development.[30][31] WAC is an affinity-based liquid chromatographic technique that separates chemical compounds based on their different weak affinities to an immobilized target. The higher affinity a compound has towards the target, the longer it remains in the separation unit, and this will be expressed as a longer retention time. The affinity measure and ranking of affinity can be achieved by processing the obtained retention times of analyzed compounds. Affinity chromatography is part of a larger suite of techniques used in chemoproteomics based drug target identification.[citation needed]

The WAC technology is demonstrated against a number of different protein targets – proteases, kinases, chaperones and protein–protein interaction (PPI) targets. WAC has been shown to be more effective than established methods for fragment based screening.[31]

History

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Affinity chromatography was conceived and first developed by Pedro Cuatrecasas and Meir Wilchek.[32][33]

References

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[edit]
Revisions and contributorsEdit on WikipediaRead on Wikipedia

Affinity chromatography

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from Grokipedia
Affinity chromatography is a liquid-phase separation technique that exploits specific, reversible interactions between a target and a complementary immobilized on a solid stationary phase to achieve highly selective isolation and purification. This method operates through an "on/off" binding mechanism, where the target binds to the ligand under mild application conditions (e.g., neutral and physiological salt concentrations) and is subsequently eluted by altering the buffer composition, such as changing , ionic , or adding a competitor, to disrupt the interaction without denaturing the . The origins of affinity chromatography trace back to the early 20th century, with foundational work by Emil Starkenstein in 1910, who demonstrated the adsorption of the enzyme α-amylase to insoluble as a means of purification. Early developments included Karl Landsteiner's 1920 experiments on antibody-antigen interactions and the 1935 use of antigen-coated charcoal by D’Alessandro and Sofia for antibody isolation, but these lacked efficient immobilization strategies. A pivotal advancement occurred in 1951 when David H. Campbell and colleagues introduced covalent attachment of to carriers like p-aminobenzylcellulose, enabling more stable biospecific adsorbents. The modern technique emerged in the 1960s, with Sven Hjerten's 1964 invention of beaded as a support matrix and the 1967 cyanogen bromide (CNBr) activation method by Ragnar Axén, Jerker Porath, and Sven Ernback, which simplified ligand immobilization. In 1968, Pedro Cuatrecasas and colleagues formalized the approach by purifying enzymes like using these innovations, coining the term "affinity chromatography" and establishing its core principles. Over the subsequent decades, the technique evolved significantly, incorporating (HPAC) in the 1970s–1980s with rigid silica supports for faster separations under higher pressures, and more recently, monolithic columns and non-biological ligands such as molecularly imprinted polymers (MIPs) and aptamers for broader applicability. Key applications span purification (e.g., enzymes, antibodies, and recombinant proteins), analytical assays like immunoextraction for monitoring, chiral separations in pharmaceuticals, and studies of biomolecular interactions, with over 122,000 mentions in as of 2019 underscoring its widespread impact in fields such as biochemistry and . In clinical contexts, it facilitates high-sensitivity testing of samples for analytes like therapeutic , hormones, and biomarkers via (HPLC)-based methods.

Fundamentals

Principle

Affinity chromatography is a bioseparation technique that exploits highly specific and reversible interactions between a target , known as the , and a complementary immobilized on a solid support matrix. This method, pioneered in its modern form by Cuatrecasas and colleagues in 1968 using beaded supports and activation for attachment, enables the selective isolation of biomolecules such as proteins, enzymes, or nucleic acids from complex mixtures. The specificity arises from biological recognition events, akin to enzyme-substrate or antigen-antibody binding, allowing purification based on affinity rather than physical properties alone. The general process involves three main stages: sample application, washing, and elution. During sample application, the mixture is introduced in a buffer that promotes binding between the and the immobilized , retaining the target while permitting unbound components to flow through. Washing follows with a buffer to remove non-specifically bound or weakly interacting materials, minimizing contamination. Elution then disrupts the interaction using competitive agents that mimic the , or by altering conditions such as , , or temperature to weaken the binding and release the purified . This technique offers key advantages over other chromatographic methods, including ion-exchange and , due to its superior selectivity, specificity, and resolution, often achieving purification factors of 100- to 10,000-fold in a single step from crude extracts. The binding equilibrium is governed by the association constant Ka=[Bound complex][Free analyte]×[Free ligand]K_a = \frac{[ \text{Bound complex} ]}{[ \text{Free analyte} ] \times [ \text{Free ligand} ]}, with the dissociation constant Kd=1KaK_d = \frac{1}{K_a} typically ranging from nanomolar (nM) to micromolar (μM) for effective separations, corresponding to KaK_a values of 10610^6 to 10910^9 M−1^{-1}. The support matrix plays a crucial role by providing an inert, porous structure to covalently attach the , ensuring minimal non-specific interactions and facilitating efficient . Common materials include for its and large pore size, silica for high-pressure stability, and magnetic beads for facile handling in batch formats. These matrices maintain the ligand's activity while supporting high binding capacities, typically 1-50 mg of protein per mL of support.

Binding Mechanisms

Affinity chromatography relies on specific, reversible interactions between a target and an immobilized , primarily driven by non-covalent forces such as hydrogen bonding, electrostatic interactions, hydrophobic effects, and van der Waals forces. These interactions mimic natural biological recognition events, allowing selective capture of the analyte from complex mixtures while maintaining the structural integrity of biomolecules like proteins or nucleic acids. The strength and specificity of these bindings are quantified by the (Kd), which typically ranges from micromolar to nanomolar values for high-affinity pairs, ensuring efficient separation under mild conditions. Ligand-analyte specificity is central to the technique, with common examples including antigen-antibody pairs, where antibodies recognize unique epitopes on antigens; enzyme-substrate or enzyme-inhibitor complexes, exploiting the active site's precise fit; and receptor-ligand interactions, such as hormone-receptor bindings. These interactions are inherently reversible, enabling the release of bound analytes through mild strategies that disrupt the non-covalent bonds without denaturation, a principle that underpins the method's . In multivalent systems, such as those involving antibodies with multiple binding sites, enhances overall binding strength beyond individual affinities, contributing to higher selectivity and retention on the column. To achieve stable immobilization, ligands are covalently attached to a solid support matrix, commonly agarose or silica beads, using activation chemistries that target functional groups like primary amines on the ligand. The cyanogen bromide (CNBr) method, introduced in the early development of affinity supports, activates hydroxyl groups on the matrix to form reactive cyanate esters that couple with amines, forming stable isourea linkages, though proper control of reaction conditions is essential to minimize ligand inactivation. N-hydroxysuccinimide (NHS) esters provide an alternative, reacting selectively with amines to yield amide bonds, which offer advantages in ligand orientation by allowing site-specific attachment near the binding site, thereby reducing steric hindrance and preserving activity. Orientation and stability are critical, as random attachment can lead to reduced accessibility and lower effective binding capacity. Binding affinity and kinetics are influenced by environmental factors, including , which modulates charge states in electrostatic interactions; , affecting the of hydrophobic effects; and , which screens electrostatic forces and impacts the Kd. For instance, buffers are often designed to approximate physiological conditions ( 7-8, moderate salt) to favor native binding conformations. Multivalent can amplify these effects, lowering the apparent Kd in clustered setups. Despite these advantages, potential pitfalls include ligand leakage, where incomplete covalent coupling or hydrolysis of linkages releases free ligand, contaminating eluates; non-specific adsorption, arising from unintended interactions with the matrix that reduce purity; and matrix effects, such as diffusion limitations or steric constraints that slow binding kinetics and lower resolution. Strategies like matrix pre-treatment and ligand spacers mitigate these issues, but they remain key considerations in method optimization.

Experimental Configurations

Batch Methods

Batch methods in affinity chromatography employ a discontinuous, stirred-tank approach for separation, where the sample is directly mixed with affinity ligands immobilized on solid supports like beads, facilitating specific binding without the need for a packed column. This technique leverages the core binding principles of reversible interactions between the target and to achieve selective capture in a simple batch format. The standard procedure starts with preparing the affinity matrix, typically ligand-coupled magnetic beads or resins suspended in a buffer compatible with the binding conditions. The sample, containing the target such as a protein, is then added to the beads in a microcentrifuge tube or similar vessel, and the mixture is gently agitated to promote contact. Incubation follows for a period sufficient for equilibrium binding, after which the beads are separated from the supernatant using a for magnetic particles or for non-magnetic resins. Unbound contaminants are removed through multiple washing steps with binding buffer, and the target is eluted by altering conditions, such as shift or competitive displacement, to disrupt the affinity interaction and release the bound . Volumes are generally small, with 10–100 μL of beads mixed with 0.5–5 mL of sample, enabling efficient processing in standard labware. Key materials include superparamagnetic beads (often 1–50 μm in diameter) coated with agarose or silica and functionalized with ligands, housed in microcentrifuge tubes or multi-well plates for parallel processing; these supports allow rapid magnetic capture without sedimentation issues. Optimization is critical for yield and purity: incubation times typically range from 5 minutes to several hours based on association kinetics, with shorter times suiting fast-binding pairs and longer for weaker affinities; bead-to-sample ratios are adjusted (e.g., 1:10 to 1:50 v/v) to match binding capacity and avoid saturation; and mixing is performed gently via end-over-end rotation or low-speed vortexing to maximize diffusion-limited contact while minimizing shear forces that could denature fragile biomolecules like enzymes or antibodies. These methods offer significant advantages, including operational simplicity with minimal equipment needs, fast turnaround times often under 1 hour per cycle, reduced costs from reusable beads and low buffer volumes, and ease of scaling for high-throughput applications like screening multiple samples in 96-well formats—making them particularly valuable for exploratory purifications or scenarios where column setup is cumbersome. Despite these benefits, batch methods exhibit limitations such as inherently lower resolution and purity compared to column-based systems, owing to the lack of directional flow that improves and zonal separation; additionally, incomplete recovery can occur with viscous or high-solid-content samples, where magnetic or centrifugal separation slows, potentially leading to carryover of impurities.

Column-Based Systems

Column-based affinity chromatography utilizes packed columns within systems such as (FPLC) or (HPLC) to enable continuous flow and high-resolution separations of biomolecules based on specific interactions. , including antibodies, enzymes, or metal ions, are covalently or non-covalently immobilized on porous supports like beads, silica, or monoliths, then slurry-packed into cylindrical columns to form a stable bed. Sample loading occurs via peristaltic or syringe pumps, introducing the mixture at controlled flow rates typically between 0.1 and 5 mL/min to ensure efficient and minimize band broadening. This setup contrasts with batch methods by providing precise flow dynamics for enhanced purity and yield in preparative applications. The operational protocol follows a standardized sequence to maximize specificity and recovery. The column is first equilibrated with a binding buffer adjusted to optimal and , promoting selective adsorption of the target analyte. During loading, the sample is passed through the bed, allowing unbound components to flow through while the target binds reversibly to the ligands. with the equilibration buffer then removes non-specifically bound impurities, followed by using either step changes—such as shifts or competitive agents—or linear gradients of salt, , or eluents to desorb the purified fraction in a concentrated peak. Regeneration concludes the cycle, involving harsh cleaning agents like to strip residual contaminants and restore the column for subsequent runs, often supporting 50–200 cycles depending on the ligand-support chemistry. Instrumentation enhances reproducibility and monitoring in these systems. Integrated platforms like the ÄKTA series from GE Healthcare automate pump control, buffer switching, and fraction collection, with inline UV detectors typically set at 280 nm to track protein elution profiles and conductivity sensors to verify buffer conditions. These features enable real-time data acquisition and method optimization, reducing manual intervention and improving throughput for both research and industrial settings. Scaling column-based systems accommodates diverse throughput needs while preserving performance. Analytical-scale columns feature small diameters (1–10 mm) and short bed heights (5–50 mm) for microgram quantities, whereas preparative scales employ larger diameters (up to 200 mm or more) and taller beds (up to several meters) to process liters of sample. Linear , often maintained at 25–150 cm/h, ensures consistent and resolution across scales; column volume scales with the square of the diameter for equal bed height, but requires proportional adjustments to avoid channeling. Separation is quantified by the height equivalent to a theoretical plate (HETP), ideally below 0.1 mm for high-performance supports, guiding support selection and packing quality. Despite advantages, column-based systems face operational hurdles that impact longevity and cost. Clogging from sample particulates or aggregates can obstruct flow paths, necessitating pre-filtration or guard columns for mitigation. buildup arises from compression under flow or viscous samples, particularly with soft supports, limiting maximum velocities and requiring rigid alternatives like silica for HPLC. stability degrades over cycles due to chemical leaching or denaturation, reducing binding capacity by 20–50% after 100 uses in some cases, while high backpressures (>10 bar) can shorten column lifespan. These challenges are addressed through expanded designs or monolithic supports to maintain low pressure drops and extended reusability.

Core Applications

Immunoaffinity Purification

Immunoaffinity purification is a specialized form of affinity chromatography that employs antibodies as immobilized ligands to selectively capture and isolate target antigens, such as proteins, peptides, viruses, or cells, based on highly specific antigen-antibody interactions. This technique leverages the natural recognition properties of antibodies, enabling purification from complex biological matrices like serum or cell lysates with exceptional selectivity. The process typically involves loading the sample onto a column or matrix with immobilized antibodies, washing away unbound components, and eluting the bound target under conditions that disrupt the interaction. Ligand preparation begins with the selection of monoclonal or specific to the target antigen, which are then immobilized on solid supports such as , silica, or magnetic beads. Covalent attachment methods, including coupling via amine, sulfhydryl, or carbohydrate groups on the , ensure stable fixation, while optimal support pore sizes of 300–500 Å facilitate efficient binding in high-performance formats. To enhance orientation and accessibility of the antigen-binding Fab regions, antibodies are often immobilized indirectly through or Protein G, which bind reversibly to the Fc region, positioning the outward and increasing binding capacity by up to twofold compared to random immobilization. This oriented approach minimizes steric hindrance and improves overall efficiency. Applications of immunoaffinity purification span and , including the isolation of specific proteins for , the production of monoclonal antibodies in therapeutics, and the depletion of abundant proteins in diagnostics. It is particularly valuable for purifying viruses or cells in development and for extracting biomarkers from clinical samples. In pharmaceutical contexts, it supports the analysis of drug-protein interactions and the purification of biologics like insulin. Elution strategies aim to release the target while preserving its , often using low pH buffers ( 2–3) to protonate key residues and weaken ionic bonds in the antigen-antibody complex. Alternative methods include high salt concentrations, chaotropic agents like or (1.5–8 M), or organic modifiers to disrupt hydrogen bonding and hydrophobic interactions. For sensitive targets, gentler competitive with excess peptides or analogs displaces the bound molecule without harsh conditions. The primary advantage of immunoaffinity purification lies in its extreme specificity, driven by dissociation constants (K_d) typically ranging from 10^{-9} to 10^{-12} M for high-affinity monoclonal , allowing near-quantitative recovery of targets from complex mixtures. This enables high purity levels, often exceeding 95%, with minimal non-specific binding. However, disadvantages include the high cost of antibody production and immobilization, as well as the risk of target denaturation during , which can reduce yields for labile biomolecules. Additionally, antibody supports may exhibit limited reusability due to potential leaching or inactivation. Representative examples include the isolation of cytokines such as tumor necrosis factor-alpha (TNF-α) from inflammatory fluids using anti-TNF-α monoclonal antibodies, achieving high specificity in hypercytokinemia studies, and the purification of hormones like insulin from pancreatic extracts or serum, where oriented immobilization enhances recovery efficiency.

Immobilized Metal Ion Affinity Chromatography

Immobilized metal ion affinity chromatography (IMAC) is a technique that exploits the coordination chemistry between ions and electron donor groups on proteins, particularly the imidazole side chains of residues, to achieve selective purification. The method relies on the immobilization of metal ions, such as Ni²⁺, Co²⁺, or Cu²⁺, onto a solid support via chelating ligands like (NTA) or (IDA), which form stable complexes with the metals while leaving coordination sites available for protein binding. This coordination occurs primarily through the nitrogen atoms in the imidazole rings of polyhistidine tags (His-tags), typically consisting of 6-10 consecutive residues engineered onto recombinant proteins, enabling high-affinity interactions under mild conditions. Originally developed for the separation of proteins with natural metal-binding properties, IMAC has become a cornerstone for purifying histidine-tagged recombinant proteins due to its simplicity, scalability, and compatibility with denaturing agents. In practice, columns or are prepared by charging the chelator-functionalized matrix with a solution of the desired metal salt, such as for Ni-NTA , a widely used commercial that provides tetradentate coordination for enhanced stability and specificity. Protein binding is performed at neutral pH (typically 7.4-8.0) in a buffer containing 10-20 mM and high salt (e.g., 0.5 M NaCl) to promote hydrophobic interactions while minimizing non-specific binding; the low concentration competes weakly with the . Washing steps follow with buffers containing 20-50 mM to remove loosely bound contaminants, and is achieved either by increasing to 200-500 mM to displace the protein-metal interaction or by chelating agents like 100-500 mM EDTA to strip the metal ions, releasing the bound protein. This process operates under nondenaturing conditions, preserving protein activity, and can be adapted for both analytical and preparative scales. IMAC offers high specificity for overexpressed recombinant proteins bearing His-tags, often achieving recoveries exceeding 90% and purities up to 95% in a single step, particularly when combined with optimized buffer compositions to reduce copurification of host cell proteins. The technique's effectiveness stems from the multivalent coordination of multiple imidazole groups, which provides dissociation constants in the micromolar range, far stronger than single-residue interactions. Variations extend its utility to non-tagged proteins containing natural histidine-rich regions, such as certain metalloproteins or phosphoproteins, though selectivity may be lower without the engineered tag. To mitigate potential metal ion contamination in the eluate, which can affect downstream applications, with EDTA or selection of softer Lewis acid metals like Co²⁺ is employed, alongside post-purification dialysis. Despite these advantages, careful optimization is required to avoid issues like resin leakage or reduced capacity from metal oxidation.

Lectin-Based Separation

Lectin-based affinity chromatography leverages the specific recognition of carbohydrate structures by —non-enzymatic proteins that bind to glycans on glycoproteins—for the selective purification and of these biomolecules. This technique is particularly valuable in separating glycoproteins based on their glycan motifs, distinguishing it from other affinity methods through its focus on carbohydrate-ligand interactions. Key lectins employed include concanavalin A (ConA), which specifically binds α-D-mannose and α-D-glucose residues found in high-mannose and hybrid N-glycans, and , which targets and residues common in complex N- and O-glycans. These lectins enable precise of glycoforms, with ConA often used for initial enrichment of mannose-rich structures and WGA for sialylated variants. In glycobiology, this method supports glycan analysis to elucidate structural diversity, purification of proteins from solubilized cell extracts, and isolation of viral glycoproteins such as those from viruses, where lectin binding exploits the dense on proteins. For instance, α-D-mannose-specific like ConA facilitate the recovery of viral , aiding production and structural studies. Binding occurs through reversible, non-covalent interactions at the lectin active sites, enhanced by multivalency where clustered glycans on the target engage multiple lectin sites, boosting overall and retention on the column. Elution is achieved primarily via with free monosaccharides, such as 0.1–0.5 M methyl-α-D-mannopyranoside for ConA-bound fractions, which competitively displaces the ; for certain lectins like , 0.1 M buffer at pH 6.5 serves as an alternative eluent to disrupt interactions. Lectins are immobilized on porous supports like (e.g., beads) to create stable affinity matrices, allowing high-capacity binding (typically 2–10 mg per mL resin) while maintaining lectin orientation for optimal accessibility. heterogeneity, arising from site-specific glycan variations, can reduce yield by causing incomplete binding of low-affinity glycoforms, often necessitating multi-lectin sequential setups for comprehensive recovery. Despite its specificity, non-specific binding to non-target carbohydrates or hydrophobic regions poses a challenge, potentially contaminating eluates; this is addressed by including deglycosylation controls (e.g., via PNGase F treatment) to confirm glycan-dependent retention and optimize washing conditions.

Boronate Affinity Techniques

Boronate affinity techniques exploit the reversible covalent bonding between ligands and cis- groups, such as vicinal diols found in sugars, , and catechols. The mechanism involves the formation of a pH-dependent , where the exists predominantly in its tetrahedral anionic form at alkaline , facilitating nucleophilic attack by the diol hydroxyl groups to create a stable five- or six-membered cyclic . This interaction is optimal at 8-9, where binding affinity is maximized, while at lower values, the equilibrium shifts toward dissociation due to of the boronate. These techniques are widely applied in the purification of biomolecules bearing cis-diol moieties, including ribonucleotides, glycoproteins, and catechols. For instance, boronate affinity chromatography enables the selective isolation of ribonucleotides and ribonucleosides from complex mixtures, leveraging the 2',3'-cis-diol in the sugar, which provides higher selectivity over deoxyribonucleotides lacking this feature. In glycoprotein purification, the method targets or other carbohydrate residues, as demonstrated in the enrichment of monoclonal antibodies and other glycoforms. Additionally, it has been employed for purifying catechol-containing siderophores from bacterial cultures, separating them from decomposition products. In research, boronate-based systems bind glucose via its cis-diol, facilitating the measurement of (HbA1c) for long-term glucose monitoring. Ligands such as phenylboronic acid are commonly immobilized on solid supports like beads or silica particles to create stationary phases for chromatography. Immobilization typically occurs via covalent attachment, such as on silica or of with , yielding stable columns suitable for repeated use. Binding modes can be static, where ligands are fixed in place for conventional , or dynamic, involving mobile ligands in formats like for , enhancing accessibility to target molecules. This setup allows for high-capacity separations under mild aqueous conditions. Elution is achieved by disrupting the boronate ester through an acidic pH shift to below 6.5, which protonates the complex and promotes hydrolysis, or by introducing competitive agents like sorbitol or mannitol that mimic cis-diols and displace bound targets. The high selectivity for RNA over DNA stems from the absence of the 2'-hydroxyl in deoxyribose, preventing effective ester formation with DNA, while RNA's ribose enables strong binding—often with retention factors 10-100 times higher for RNA. Boronate affinity materials offer advantages including across multiple cycles, with agarose-based columns retaining over 90% binding capacity after 50-100 uses, and reusability without significant leaching. Their pH-switchable nature supports gentle conditions that preserve activity. Emerging applications extend to biosensors, where boronate-functionalized surfaces enable real-time detection of glycoproteins or glucose, as in electrochemical or surface-enhanced platforms for point-of-care diagnostics. These developments build on the seminal 1970 report by Weith et al., which first demonstrated boronate separation of and sugars.

Specialized and Emerging Uses

Recombinant Protein Isolation

Affinity chromatography plays a central role in the isolation of recombinant proteins by leveraging genetically engineered affinity tags that enable specific binding to immobilized ligands. Common tagging systems include the , typically consisting of 6-10 consecutive histidine residues that bind to ions such as or in immobilized metal affinity chromatography (); glutathione S-transferase (GST), a 26 kDa that specifically interacts with glutathione-sepharose; , which binds to resin and also enhances protein solubility; and the , a short sequence (e.g., WSHPQFEK) that has high affinity for or engineered streptactin variants. These tags are fused to the target protein during genetic construction, often with a cleavage site inserted between the tag and the protein of interest to allow tag removal post-purification, such as the tobacco etch virus (TEV) protease site (ENLYFQ/G or /S), which enables site-specific cleavage with high efficiency and minimal off-target activity. The typical workflow for recombinant protein isolation begins with expression of the tagged protein in heterologous hosts, such as for bacterial systems, yeast like Pichia pastoris, or mammalian cells like HEK293 for eukaryotic modifications. Following induction of expression, cells are harvested and lysed using mechanical (e.g., ) or chemical methods to release the intracellular contents, producing a crude lysate that is clarified by or . The lysate is then loaded onto an affinity column equilibrated with binding buffer; for instance, His-tagged proteins are captured on Ni-NTA resin under mildly denaturing conditions if necessary, while GST- or MBP-tagged proteins bind to or columns under native conditions, achieving high specificity and often >95% purity in a single step. Elution is performed using competitive agents like imidazole for His-tags, for GST, or maltose for MBP, followed by optional tag cleavage with and a secondary polishing step if required. Optimization of the tagging strategy is crucial for maximizing , yield, and functionality. The position of the tag—N-terminal versus C-terminal—can significantly influence outcomes; N-terminal fusions often improve for proteins prone to aggregation by acting as chaperones, while C-terminal tags may be preferred to avoid interference with N-terminal processing signals, though empirical testing is essential as effects vary by protein. Typical yields from bacterial cultures range from 1-10 mg of purified protein per liter, depending on expression levels and tag , with MBP frequently boosting yields by 2-5 for challenging proteins. Despite these advantages, challenges persist, including the formation of in prokaryotic hosts like E. coli, where misfolded proteins aggregate and require denaturation-refolding protocols that can reduce recovery; potential of tags in therapeutic applications, necessitating their removal to minimize immune responses; and scale-up to industrial bioreactors, where maintaining tag binding under high-density cultures demands robust stability and process controls. Emerging advancements include self-cleaving affinity tags, such as intein-based systems in resins like Cytiva Protein Select introduced in 2024, which enable automatic tag removal during purification without additional steps, improving efficiency for large-scale therapeutic . These methods have been instrumental in producing therapeutic recombinant proteins, such as insulin variants expressed in E. coli with His-tags for initial capture via before final processing, and monoclonal antibodies in mammalian systems where Strep- or His-tags facilitate early-stage purification alongside affinity steps.

Serum Protein Purification

Affinity chromatography enables the selective isolation of specific proteins from serum or plasma, which are complex biological fluids dominated by high-abundance components like (approximately 50-60% of total protein) and immunoglobulins. This technique leverages biospecific interactions to achieve high purity and recovery, facilitating downstream analyses in research and clinical settings. Common targets include , immunoglobulins, and , with procedures often employing batch or column formats to handle the viscous nature of serum samples. Albumin purification typically utilizes dye-ligand affinity chromatography with Cibacron Blue F3GA immobilized on matrices like agarose or magnetic particles, exploiting the dye's structural mimicry of NAD and other dinucleotides to bind albumin's hydrophobic pockets at neutral pH. Serum is diluted and loaded onto the column, followed by washing with low-salt buffers and elution using high salt concentrations (e.g., 1 M NaCl) or competitive ligands like ATP, yielding purities exceeding 95% and adsorption capacities around 48-50 mg/g in optimized systems. For immunoglobulins, particularly IgG, Protein A or Protein G ligands are employed, binding the Fc region with dissociation constants in the micromolar range; this allows single-step purification to near homogeneity (>95% purity) from serum via pH-based elution (e.g., glycine-HCl at pH 3). Transferrin, a metal-binding glycoprotein, is isolated using immobilized metal affinity chromatography (IMAC) with chelated ions such as Cu²⁺ or Zn²⁺, which coordinate with its histidine residues, enabling separation from serum under mild conditions as demonstrated in early metal chelate protocols. A variation involves heparin affinity chromatography for coagulation factors like antithrombin III and factor Xa, where immobilized heparin binds these proteins with high specificity (K_d ~10-100 nM), aiding purification for therapeutic applications. To mitigate proteolytic degradation during processing, protease inhibitors such as PMSF or EDTA are routinely added to lysis and elution buffers. These methods find broad applications in clinical diagnostics, where purified serum proteins serve as biomarkers (e.g., for iron status assessment) or reagents in assays, and in biomarker discovery through workflows. A key use is the depletion of abundant proteins like and IgG via immunoaffinity columns, which removes up to 85-90% of total serum protein mass, enhancing detection of low-abundance candidates by 10- to 20-fold in mass spectrometry-based analyses. Such depletions improve proteomic depth without compromising sample integrity, supporting studies on disease-specific alterations in serum profiles.

Weak Affinity Chromatography

Weak affinity chromatography (WAC) represents an adaptation of traditional affinity chromatography tailored for the study and separation of low-affinity interactions, typically those with dissociation constants (K_d) greater than 1–10 μM, enabling the analysis of transient biomolecular bindings that are often overlooked in high-affinity methods. This approach leverages reversible, weak biospecific recognition between immobilized ligands and , often under isocratic conditions without the need for harsh buffers, making it particularly suitable for equilibrium-based measurements of binding kinetics. Frontal affinity chromatography (FAC), a key variant, continuously applies the to the column until a breakthrough curve is observed, quantifying weak interactions through the retardation of analyte migration, while zonal WAC injects discrete samples and assesses retention or peak broadening for similar purposes. These methods are especially valuable in drug screening, where they facilitate the evaluation of low-affinity ligands that may evolve into potent inhibitors. The experimental setup for weak affinity chromatography emphasizes conditions that promote equilibrium binding without overwhelming non-specific interactions, typically involving low densities on the stationary phase (e.g., or supports) to avoid saturation and ensure reversibility. Flow rates are kept slow, often in the range of 0.01–0.1 mL/min, to allow sufficient for weak associations, particularly in miniaturized columns that enhance sensitivity for precious samples like proteins in nanodiscs. Analysis in FAC relies on breakthrough curves, where the volume at half-height of the curve provides binding parameters, whereas zonal WAC examines peak broadening or retention factors to derive kinetic , often coupled with for identification. These configurations minimize limitations, enabling accurate characterization of interactions with association constants (K_a) below 10^5–10^6 M^{-1}. In applications, weak affinity chromatography excels in fragment-based drug discovery, where small molecular fragments with micromolar affinities are screened against targets like kinases or G-protein coupled receptors, identifying hits that can be optimized into leads. It is also widely used for probing protein-protein interactions, such as those involving and glycans, providing insights into transient complexes critical for cellular signaling. Results from WAC are frequently validated through orthogonal techniques like (SPR) or (ITC), which confirm binding affinities and kinetics derived from chromatographic data. For instance, in screening libraries of up to 1,000 fragments, WAC has successfully ranked binders by retention times, with subsequent SPR validation yielding K_d values in the 10–200 μM range. The mathematical foundation of weak affinity chromatography is grounded in the Langmuir adsorption isotherm, which models the fractional occupancy (θ) of binding sites as θ = (K_a × C) / (1 + K_a × C), where K_a is the association constant and C is the concentration; for weak binding, this simplifies to linear approximations where retention is proportional to K_a. This framework allows derivation of K_d from experimental observables like retention factor k ≈ (B_t / V_m) × K_a, with B_t as active binding sites and V_m as void volume, ensuring quantitative analysis even at low affinities. Weak affinity chromatography offers distinct advantages in detecting transient interactions that evade capture in strong-affinity systems, providing rapid, with minimal sample consumption and no need for immobilization of the target analyte. However, challenges include suboptimal signal-to-noise ratios due to weak retention, necessitating optimized column capacities and of non-specific binding contributions to achieve reliable data.

Historical Development

Origins and Invention

Affinity chromatography emerged in the late amid a post-World War II surge in biochemical research, which emphasized techniques and paralleled developments in gel filtration chromatography introduced by Porath and Flodin in 1959 and ion-exchange methods refined in the . This era saw rapid advancements in separation science, driven by the need to isolate enzymes and biomolecules for studying metabolic pathways and molecular interactions, building on earlier adsorption-based approaches. Earlier foundations include Emil Starkenstein's 1910 work on adsorbing α-amylase to insoluble . Early foundations for affinity chromatography stemmed from immobilization studies, notably the 1916 work by Nelson and Griffin, who demonstrated the adsorption of onto and aluminum , retaining enzymatic activity for . This biospecific adsorption concept influenced later techniques. A critical advance came from Porath and colleagues in 1967, who developed (CNBr) activation of for covalent attachment of proteins and ligands, enabling stable immobilization of bioactive molecules onto solid supports. This built on Hjerten's 1964 development of beaded () as a support matrix. The technique was formalized and named by Pedro Cuatrecasas, who, in collaboration with Meir Wilchek and , described affinity chromatography in 1968 as a method for selective enzyme purification using immobilized substrates. In their seminal PNAS publication, they purified staphylococcal by passing crude extracts through columns containing the specific inhibitor thymidine 3',5'-bisphosphate (pdTp) covalently bound to , achieving up to 500-fold purification in a single step via specific reversible binding to the enzyme's . Similar applications included purification of dehydrogenases using immobilized NAD. This column-based format marked a shift from prior batch adsorption methods, improving efficiency and scalability. Initial implementations faced challenges with ligand stability, as bound substrates could leach under elution conditions, and support materials like early cellulose derivatives lacked sufficient mechanical strength for high-flow columns. Cuatrecasas and team addressed these by adopting beaded () activated via the Porath CNBr method, which provided better flow properties and reduced non-specific binding.

Key Advancements

In the , a pivotal advancement came with the introduction of immobilized metal affinity chromatography (IMAC) by Jerker Porath and colleagues, who demonstrated the use of chelated transition metals like and to selectively bind histidine-rich proteins, enabling efficient purification without harsh conditions. Concurrently, the development of lectin-based affinity columns, leveraging carbohydrate-binding proteins such as concanavalin A immobilized on , facilitated the separation of glycoproteins, laying groundwork for glycoproteomics applications. The and saw the rise of technologies enabling the widespread adoption of polyhistidine (His) tags for , first described by Hochuli et al. in 1987 using nickel-chelated resins, which simplified IMAC for overexpressed proteins in . Commercialization accelerated during this period, with companies like (formerly ) introducing standardized kits such as Ni-NTA in the early , making affinity methods accessible for routine lab use and industrial-scale production. By the 2000s, affinity chromatography integrated with high-throughput formats and , allowing automated parallel purifications in microscale devices for faster screening, as exemplified in workflows. A major leap was its coupling with , enabling direct analysis of affinity-enriched peptides for large-scale , with techniques like stable labeling enhancing quantification in studies of protein interactions. Post-2010 developments have focused on nanomaterial supports, such as functionalized with affinity , which enable rapid, magnetically driven separations reducing processing time from hours to minutes while maintaining high specificity for biomolecules. Single-use affinity systems, including disposable columns and membranes, gained traction in manufacturing to minimize cross-contamination and costs, particularly for production. AI-driven approaches to design, using to predict and optimize binding affinities, have emerged to create more selective and stable sorbents for challenging targets. Expansions into involve boronate or aptamer-based affinities for small-molecule isolation, while exosome purification employs antibody-conjugated beads for extracellular vesicle enrichment in diagnostic applications. Key milestones include the 2018 awarded to Frances H. Arnold for , which has been instrumental in engineering novel affinity tags and ligands for enhanced selectivity in . The accelerated immunoaffinity methods, with isolations using Ni-NTA or anti-spike antibodies on resins, supporting development and serological testing at scale.

References

  1. https://pmc.ncbi.nlm.nih.gov/articles/PMC7584770/
  2. https://digitalcommons.unl.edu/cgi/viewcontent.cgi?article=1075&context=chemistryhage
  3. https://doi.org/10.1073/pnas.61.2.636
  4. https://pmc.ncbi.nlm.nih.gov/articles/PMC3578177/
  5. https://pmc.ncbi.nlm.nih.gov/articles/PMC5523865/
  6. https://www.sciencedirect.com/science/article/pii/S0021967322007257
  7. https://doi.org/10.1038/2141302a0
  8. https://www.frontiersin.org/journals/molecular-biosciences/articles/10.3389/fmolb.2020.615565/full
  9. https://pmc.ncbi.nlm.nih.gov/articles/PMC544596/
  10. https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-biology-learning-center/protein-biology-resource-library/pierce-protein-methods/overview-affinity-purification.html
  11. https://pmc.ncbi.nlm.nih.gov/articles/PMC6617771/
  12. https://documents.thermofisher.com/TFS-Assets/LSG/Application-Notes/TR0004-Batch-affinity.pdf
  13. https://www.iba-lifesciences.com/applications/protein-purification/magnetic-beads-purification/
  14. https://pmc.ncbi.nlm.nih.gov/articles/PMC10287800/
  15. https://www.sciencedirect.com/science/article/pii/S0021967324005752
  16. https://pmc.ncbi.nlm.nih.gov/articles/PMC7513800/
  17. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7584770/
  18. https://home.sandiego.edu/~josephprovost/ProteinBookletFull.pdf
  19. https://pmc.ncbi.nlm.nih.gov/articles/PMC2903764/
  20. https://pmc.ncbi.nlm.nih.gov/articles/PMC3346860/
  21. https://pmc.ncbi.nlm.nih.gov/articles/PMC5374010/
  22. https://www.sciencedirect.com/science/article/abs/pii/S0021967321006026
  23. https://pmc.ncbi.nlm.nih.gov/articles/PMC2909483/
  24. https://pmc.ncbi.nlm.nih.gov/articles/PMC8585745/
  25. https://www.sciencedirect.com/topics/neuroscience/concanavalin-a
  26. https://www.sciencedirect.com/science/article/pii/S0006497120844814
  27. https://digitalcommons.unl.edu/cgi/viewcontent.cgi?article=1064&context=chemistryhage
  28. https://pubmed.ncbi.nlm.nih.gov/391282/
  29. https://pubmed.ncbi.nlm.nih.gov/17011087/
  30. https://www.sciencedirect.com/science/article/abs/pii/S0264410X06009947
  31. https://glycopedia.eu/echapter/article-how-do-lectin-read-the-glyco-code/article-understanding-multivalency/
  32. https://www.frontiersin.org/journals/molecular-biosciences/articles/10.3389/fmolb.2021.746822/full
  33. https://cdn.cytivalifesciences.com/api/public/content/digi-17674-pdf
  34. https://www.gbiosciences.com/Immobilized-Lectins
  35. https://pubmed.ncbi.nlm.nih.gov/16555004/
  36. https://pmc.ncbi.nlm.nih.gov/articles/PMC3703587/
  37. https://pubs.acs.org/doi/10.1021/acs.accounts.7b00179
  38. https://www.researchgate.net/publication/283072582_Boronate_Affinity_Chromatography
  39. https://experiments.springernature.com/articles/10.1007/978-1-60327-261-2_12
  40. https://pubmed.ncbi.nlm.nih.gov/10420578/
  41. https://digitalcommons.unl.edu/context/chemistryhage/article/1064/viewcontent/Hage_CC_1999_Affinity_Chromatography.pdf
  42. https://pmc.ncbi.nlm.nih.gov/articles/PMC2212591/
  43. https://www.researchgate.net/publication/6958629_Boronic_Acids_as_Ligands_for_Affinity_Chromatography
  44. https://www.bio-rad.com/webroot/web/pdf/lsr/literature/Bulletin_1066B.pdf
  45. https://www.sciencedirect.com/science/article/abs/pii/S0021967398010802
  46. https://pmc.ncbi.nlm.nih.gov/articles/PMC10452607/
  47. https://pmc.ncbi.nlm.nih.gov/articles/PMC4527311/
  48. https://bitesizebio.com/48730/affinity-tag/
  49. https://pmc.ncbi.nlm.nih.gov/articles/PMC7974378/
  50. https://www.neb.com/en-us/products/p8112-tev-protease
  51. https://www.med.unc.edu/pharm/sondeklab/wp-content/uploads/sites/868/2018/10/Recombinant-protein-purification-handbook.pdf
  52. https://analyticalsciencejournals.onlinelibrary.wiley.com/doi/10.1002/biot.201100155
  53. https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2014.00063/full
  54. https://pmc.ncbi.nlm.nih.gov/articles/PMC7125906/
  55. https://www.frontiersin.org/journals/bioengineering-and-biotechnology/articles/10.3389/fbioe.2021.630551/full
  56. https://pmc.ncbi.nlm.nih.gov/articles/PMC7902521/
  57. https://www.insightaceanalytic.com/report/advanced-chromatographic-solutions-for-biomolecules-market/3246
  58. https://www.technologynetworks.com/biopharma/articles/therapeutic-proteins-types-design-and-production-404135
  59. https://pmc.ncbi.nlm.nih.gov/articles/PMC2958570/
  60. https://pmc.ncbi.nlm.nih.gov/articles/PMC5241407/
  61. https://pmc.ncbi.nlm.nih.gov/articles/PMC10760827/
  62. https://pmc.ncbi.nlm.nih.gov/articles/PMC2517482/
  63. https://pmc.ncbi.nlm.nih.gov/articles/PMC4621480/
  64. https://pubmed.ncbi.nlm.nih.gov/21400175/
  65. https://pubmed.ncbi.nlm.nih.gov/16052620/
  66. https://pubmed.ncbi.nlm.nih.gov/16212426/
  67. https://www.sciencedirect.com/science/article/pii/S0304416520302725
  68. https://www.mdpi.com/1420-3049/28/20/7113
  69. https://pmc.ncbi.nlm.nih.gov/articles/PMC10608817/
  70. https://www.nature.com/articles/1831657a0
  71. https://pubs.acs.org/doi/10.1021/ja02262a018
  72. https://www.nature.com/articles/2151491a0
  73. https://www.pnas.org/doi/10.1073/pnas.61.2.636
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