Various methods are used to enrich or purify a protein of interest from other proteins and components in a crude cell lysate or other sample. The most powerful of these methods is affinity chromatography, also called affinity purification, whereby the protein of interest is purified by virtue of its specific binding properties to an immobilized ligand.

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Pierce Protein Purification
Technical Handbook

Introduction

Affinity Purification vs. Other Methods

Proteins and other macromolecules of interest can be purified from crude extracts or other complex mixtures by a variety of methods. Precipitation is perhaps the simplest method for separating one type of macromolecule from another. For example, nucleic acids can be precipitated and thereby purified from undesired molecules in solution using ethanol. Similarly proteins can be selectively precipitated in the presence of acetone.

Most purification methods, however, involve some form of chromatography whereby molecules in solution (mobile phase) are separated based on differences in chemical or physical interaction with a stationary material (solid phase). Gel filtration (also called size-exclusion chromatography or SEC) uses a porous resin material to separate molecules based on size; large molecules are excluded from the tiny internal spaces of the resin beads while small molecules enter the resin pores, resulting in a longer path through the column. In ion exchange chromatography, molecules are separated according to the strength of their overall ionic interaction with a solid phase material; by manipulating buffer conditions, molecules of greater or lesser ionic character can be bound to or dissociated from the solid phase material.

By contrast with the aforementioned methods, affinity chromatography (also called affinity purification) makes use of specific binding interactions between molecules. A particular ligand is chemically immobilized or “coupled” to a solid support so that when a complex mixture is passed over the column, those molecules having specific binding affinity to the ligand become bound. After other sample components are washed away, the bound molecule is stripped from the support, resulting in its purification from the original sample.

Each specific affinity system requires its own set of conditions and presents its own peculiar challenges for a given research purpose. Other Protein Methods articles describe the factors and conditions associated with particular purification systems (see links in side bar near the end of this page). Nevertheless, the general principles involved are the same for all ligand-target binding systems, and these concepts are the focus of this overview.

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Gel filtration (desalting)

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Tech Tip #49: Acetone precipitation of proteins.

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Desalting Columns
Ion Exchange Columns

How Affinity Purification Works

Affinity purification generally involves the following steps:

1. Incubate crude sample with the affinity support to allow the target molecule in the sample to bind to the immobilized ligand.
2. Wash away nonbound sample components from the support.
3. Elute (dissociate and recover) the target molecule from the immobilized ligand by altering the buffer conditions so that the binding interaction no longer occurs.

A single pass of a serum or cell-lysate sample through an affinity column can achieve greater than 1,000-fold purification of a specific protein so that only a single band is detected after gel electrophoresis (e.g., SDS-PAGE) analysis.

Ligands that bind to general classes of proteins (e.g., antibodies) or commonly used fusion protein tags (e.g., 6xHis) are commercially available in pre-immobilized forms ready to use for affinity purification. Alternatively, more specialized ligands such as specific antibodies or antigens of interest can be immobilized using one of several commercially available activated affinity supports; for example, a peptide antigen can be immobilized to a support and used to purify antibodies that recognize the peptide.

Most commonly, ligands are immobilized or “coupled” directly to solid support material by formation of covalent chemical bonds between particular functional groups on the ligand (e.g., primary amines, sulfhydryls, carboxylic acids, aldehydes) and reactive groups on the support (see related article on Covalent Immobilization). However, indirect coupling approaches are also possible. For example, a GST-tagged fusion protein can be first captured to a glutathione support via the glutathione-GST affinity interaction and then secondarily chemically crosslinked to immobilize it. The immobilized GST-tagged fusion protein can then be used to affinity purify binding partner(s) of the fusion protein.

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Covalent Immobilization

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Supports for Covalent
Protein Immobilization

Binding and Elution Buffers for Affinity Purification

Most affinity purification procedures involving protein:ligand interactions use binding buffers at physiologic pH and ionic strength, such as phosphate buffered saline (PBS). This is especially true when antibody:antigen or native protein:protein interactions are the basis for the affinity purification. Once the binding interaction occurs, the support is washed with additional buffer to remove nonbound components of the sample. Nonspecific (e.g., simple ionic) binding interactions can be minimized by adding low levels of detergent or by moderate adjustments to salt concentration in the binding and/or wash buffer. Finally, elution buffer is added to break the binding interaction and release the target molecule, which is then collected in its purified form. Elution buffer can dissociate binding partners by extremes of pH (low or high), high salt (ionic strength), the use of detergents or chaotropic agents that denature one or both of the molecules, removal of a binding factor or competition with a counter ligand. In most cases, subsequent dialysis or desalting is required to exchange the purified protein from elution buffer into a more suitable buffer for storage or downstream analysis.

The most widely used elution buffer for affinity purification of proteins is 0.1M glycine•HCl, pH 2.5-3.0. This buffer effectively dissociates most protein:protein and antibody:antigen binding interactions without permanently affecting protein structure. However, some antibodies and proteins are damaged by low pH, so eluted protein fractions should be neutralized immediately by addition of 1/10th volume of alkaline buffer such as 1M Tris•HCl, pH 8.5. Other elution buffers for affinity purification of proteins are listed the accompanying table. We offer several preformulated binding and elution buffers designed for affinity purification involving antibodies.

Supports

Solid Supports for Affinity Purification

Affinity purification involves the separation of molecules in solution (mobile phase) based on differences in binding interaction with a ligand that is immobilized to a stationary material (solid phase). A support or matrix in affinity purification is any material to which a biospecific ligand is covalently attached. Typically, the material to be used as an affinity matrix is insoluble in the system in which the target molecule is found. Usually, but not always, the insoluble matrix is a solid. Hundreds of substances have been described and utilized as affinity matrices.

Useful affinity supports are those with a high surface-area to volume ratio, chemical groups that are easily modified for covalent attachment of ligands, minimal nonspecific binding properties, good flow characteristics and mechanical and chemical stability. When choosing an affinity support or matrix for any separation, perhaps the most important question to answer is whether a reliable commercial source exists for the desired matrix material in the quantities required.

Immobilized ligands or activated affinity support chemistries are available for use in several different formats. Most commonly, crosslinked beaded agarose or polyacrylamide resins are used for column- or small-scale purification procedures. Magnetic particles to which affinity ligands have been immobilized are especially useful for cell separations and certain automated purification procedures. Even polystyrene microplates, more commonly used for assay purposes, can be used as the support for immobilizing ligands to purify binding partners.

Porous Gel Supports

Porous gel supports generally provide the most useful properties for affinity purification of proteins. These types of supports are usually sugar- or acrylamide-based polymer resins that are produced in solution (i.e., hydrated) as 50-150µm diameter beads. The beaded format allows these resins to be supplied as wet slurries that can be easily dispensed to fill and "pack" columns with resin beds of any size. The beads are extremely porous and large enough that biomolecules (proteins, etc.) can flow as freely into and through the beads as they can between and around the surface of the beads. Ligands are covalently attached to the bead polymer (external and internal surfaces) by various means. The result is a loose matrix in which sample molecules can freely flow past a high surface area of immobilized ligand.

By far the most widely used matrix for protein affinity purification techniques is crosslinked beaded agarose, which is typically available in 4% and 6% densities. (This means that a 1 ml resin-bed is more than 90% water by volume.) We also offer affinity products in a durable polyacrylamide-based, beaded resin called UltraLink Biosupport. Characteristics of these resins are listed in the accompanying table. Beaded agarose is good for routine applications but crushes easily, making it suitable for gravity-flow, low-speed-centrifugation, and low-pressure procedures. On the other hand, UltraLink Biosupport does not compress and may be used in medium pressure applications with a peristaltic pump or other liquid chromatography systems. Both supports have generally low nonspecific binding characteristics; nevertheless, they behave slightly differently in particular applications.

Magnetic Particles

Magnetic particles are a completely different type of affinity support from beaded agarose and other porous resins. They are much smaller (typically 1-4µm diameter) and solid (non-porous). Their small size is provides the sufficient surface area-to-volume ratio needed for effective ligand immobilization and affinity purification. Magnetic beads are produced as superparamagnetic iron oxide particles that are covalently coated with silane derivatives. The coating makes the beads inert (i.e., to minimize nonspecific binding) and provides the particular chemical groups needed for attaching ligands of interest.

Affinity purification with magnetic particles is not performed in column. Instead, a few microliters of beads is mixed with several hundred microliters of sample as a loose slurry. During mixing, the beads remain suspended in the sample solution, allowing affinity interactions to occur with the immobilized ligand. After sufficient time for binding has been given, the beads are collected and separated from the sample using a powerful magnet. Typically, simple bench-top procedures are done in microcentrifuge tubes, and pipetting or decanting is used to remove the sample (or wash solutions, etc.) while the magnetic beads are held in place at the bottom or side of the tube with a suitable magnet.

However, the particular advantage of magnetic particles over porous resins is their suitability for high-throughput automation. Increasingly sophisticated and powerful sample-handling instruments are available for performing assays and purification procedures using magnetic separations. Another advantage of magnetic particles is that, unlike porous resins, they can be used for cell separation procedures.

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Automated magnetic
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MagnaBind Magnetic Beads

Types of Affinity Purification

Different classes of affinity targets, as well as different purification goals, require consideration of different priorities (e.g., high purity vs. high yield), technical limitations and buffer conditions for development of a successful procedure. These different systems are described in more detail in other Protein Methods articles. Here is a brief list and description of these affinity purification systems.

Antibody Purification

Several methods of antibody purification involve affinity purification techniques. Typical laboratory-scale antibody production involves relatively small volumes of serum, ascites fluid or culture supernatant. Depending on how the antibody will be used for various assay and detection methods, it must be partially or fully purified. Three levels of purification specificity include the following approaches:

• Precipitation with ammonium sulfate. This simple technique provides crude purification of total immunoglobulin from other serum proteins.
• Affinity purification with immobilized Protein A, G, A/G or L. These proteins bind to most species and subclasses of IgG, the most abundant type of immunoglobulin produced by mammals in response to immunogens. Ready-to-use resins and purification kits with these proteins are available in many package sizes and formats.
• Affinity purification with immobilized antigen. Covalently immobilizing purified antigen (i.e., the peptide or hapten used as the immunogen to induce production of antibody by the host animal) to an affinity support allows the specific antibody to be purified from crude samples. Activated resins and complete kits for preparing immobilized antigens via a variety of chemistries are available.

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Overview of antibody purification

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Antibody Purification Products

Antigen Purification Using Antibodies

Specific antibodies are most frequently used to detect antigens of interest in assays, but they also can be used to purify antigens. Because specific antibodies are costly to produce or obtain commercially, this approach is seldom used for large scale purification of antigen. Instead, its use is confined almost entirely to very small-scales, most significantly for immunoprecipitation assays (seen next section).

Nevertheless, when purified antibody is available, it can be covalently immobilized to beaded agarose or other affinity support by any one of several efficient conjugation chemistries. Covalent immobilization via primary amines, as with our AminoLink Plus Coupling Kits, is an especially simple and effective method for preparing an antibody affinity column.

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Covalent immobilization

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AminoLink Plus Coupling Kits

Immunoprecipitation and Co-immunoprecipitation

Immunoprecipitation (IP) refers to the small-scale affinity purification of antigen using a specific antibody. Traditional immunoprecipitation involves capturing an antibody-antigen complex with immobilized Protein A or G agarose resin (Protein A or G binds the antibody, which is bound to its antigen), and then recovering the purified antigen in sample loading buffer for gel electrophoresis.

Co-Immunoprecipitation (Co-IP) involves attempting to capture and detect not only the direct antigen but also any proteins in the mileu of the cellular lysate that are interacting (i.e., bound to) the antigen. In the traditional format with Protein A or Protein G, this purification scheme involves no less than three levels of affinity interaction.

By adapting and optimizing other methods of antibody immobilization for the small scale needed for IP and Co-IP, several innovations have been developed that overcome many limitations and complications associated with traditional IP techniques.

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Immunoprecipitation
(overview and selection guide)

Co-Immunoprecipitation

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Pierce Co-IP Kit

Pull-Down Assays

Like co-immunoprecipitation, pull-down assays are an affinity approach often used for studying protein-protein interactions. However, unlike an IP or Co-IP, pull-down does not involve using an antibody specific to the target protein being studied. The minimal requirement for a pull-down assay is the availability of a purified and tagged protein (the bait) that is used to ‘pull-down’ a protein-binding partner (the prey). The bait protein is created through cloning and expression of a fusion protein or as a covalent modification, such as the addition of a biotin tag (see next two topics). The tagged (e.g., biotinylated) bait protein can be immobilized on a tag-specific affinity support (e.g., streptavidin). Immobilized bait protein is then incubated with a protein solution expressing protein(s) (the prey) that may bind to the bait. These bait-prey protein complexes can then be identified. Alternatively, activated supports can be used to directly immobilize almost any bait molecule.

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Pull-down assays

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Fusion Tag Protein Purification

When proteins are expressed recombinantly, additional amino acids, an entire domain or a whole protein is often added to aid in the purification and manipulation of the protein. These additions to a protein are known as fusion tags and are added to the DNA that encodes the native protein sequence. One of the most common fusion tags is a short string of six to nine histidine residues (known as the 6xHis or polyHis tag), which will bind to metal ions such as nickel or cobalt. Another fusion tag is glutathione S-transferase (GST), which binds tightly to reduced glutathione.

The properties of fusion tags allow tagged proteins to be manipulated easily in the laboratory. Most signficantly, the well-characterized tag-ligand chemistry enables single-step affinity purification of tagged molecules using immobilized versions of their corresponding ligands. In addition, antibodies to fusion tags are also available and can be used for "universal" purification and detection of tagged proteins (i.e., without having to obtain or develop a probe for each specific recombinant protein).

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GST-tagged proteins
His-tagged proteins

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Fusion Protein Purification

Avidin-Biotin Systems

Biotin, also known as vitamin H, is a small molecule (MW 244.3) that is present in tiny amounts in all living cells. The valeric acid side chain of the biotin molecule can be derivatized to incorporate various reactive groups that are used to attach biotin to other molecules. Once biotin is attached to a molecule, the molecule can be affinity purified because several proteins, namely avidin and streptavidin, are known to bind strongly and specifically to the biotin group.

Avidin and streptavidin proteins are readily available in native and recombinant purified forms. Together, biotin and variants of avidin make an extremely powerful affinity systems for adaptation to many kinds of research applications. Streptavidin agarose resins (immobilized streptavidin supports) enable purification or secondary immobilization of biotin-labeled proteins.

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Avidin-biotin affinity systems

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Biotin-Binding Affinity Supports

Class Enrichment and Isolation

In addition to affinity supports and ligands that allow purification of very specific targets (e.g., particular antigens or engineered tags), certain kinds of ligands enable general enrichment or isolation of certain classes of biological molecules. Protein A and Protein G, discussed above, can be thought of as example of this type of affinity system, as they bind to general classes of immunoglobulins. Generally, any unique chemical property or functional group shared by all members of a target set of molecules can become the basis for an enrichment or isolation scheme if a suitable affinity ligand can be identified.

Post-translational modifications (PTM) are good examples of such functional groups that define otherwise unrelated set of molecules. Whether it be phosphorylation, glycosylation or ubiquitination, the PTM has chemical properties that are only subtlely distinguishable from other chemical groups by most know chemical ligands. Thus any affinity system can, at best, only enrich for the target class of compounds.

Other examples include In addition to the few affinity supports whose ligands have broad application to many different protein methods, there are many others whose applications are more narrowly defined or are incorporated into kit structures for very specific purposes.

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Overview of post-translational modification (PTM)

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PTM Enrichment and
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Contaminant Removal

In some cases, the goal of affinity purification is to remove a particular class of undesirable sample components rather than to purify one target molecule. In this sense, the only difference between contaminant removal and traditional affinity purification is that one wishes to keep the flow-through sample and to throw away the bound molecule. In such as scenario, the concern is the binding buffer be suitable for sample recovery.

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Contaminant Removal Kits