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Protein Isolation

Protein isolation has become one of the most important technique in modern biochemistry studies. Traditional protein isolation strategies use chromatography and dialysis, which are laborious and protracted. Small yields, low product purities and a high degree of sample degradation are commonly found in these techniques. These drawbacks constrained their application in biochemical studies, which provide limited crude samples and require high protein purities and high remaining biological activities.

Immunoprecipitation

Beads based immunoprecipitation was developed to extract proteins of interest from a limited quantity of sample. By capturing the target proteins using their antibodies, these proteins can be immobilized onto beads for the further isolation and purification.

Schematic illustration of immunoprecipitationFig 1. Schematic illustration of immunoprecipitation

Sepharose and agarose beads are the earliest commercialized resins for immunoprecipitation. However, due to their porous structure, unspecific binding and insufficient elution are commonly found, resulting in incomplete removal of impurities and sample loss. Meanwhile, sepharose and agarose bead-based immunoprecipitation requires multiple centrifugations, which is not suitable for automated low- to high-throughput procedures.

Magnetic beads show a fast-growing trend in the application of immunoprecipitation. Comparing to sepharose and agarose beads, magnetic beads are characterized by its uniformed shape and size, low unspecific molecule absorption and rapid separation from mixtures at the presence of a magnetic field. Thus, magnetic bead-based biomolecule separation is capable of isolating target proteins with large yield and high purity directly from the crude samples. Magnetic separation required no centrifugation, and many manual magnetic separators and automated systems for magnetic particle handling and liquid handling have been commercialized.

Antibody Conjugation Strategies

  • Protein A/G

Protein A and protein G are bacterial proteins with high antibody binding affinity. They are capable of recognizing and specifically bind to the heavy chains in the Fc region of antibodies, which effectively orients the immobilized antibodies with antigen-binding sites facing outward.

Schematic illustration of protein A/G/L-mediated antibody conjugation Fig 2. Schematic illustration of protein A/G/L-mediated antibody conjugation

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Absolute Mag™ Protein A Magnetic Particles
Absolute Mag™ Protein G Magnetic Particles

  • Streptavidin-biotin binding

Due to streptavidin’s strong non-covalent binding to biotin, streptavidin-coupled beads are widely used for the immobilization of biotinylated molecules to their surface. Readily available reagents or kits have been commercialized to help the researchers biotinylated their primary antibodies in the laboratory. Meanwhile, many biotinylated primary antibodies are also commercially available.

Schematic illustration of streptavidin-biotin interaction-mediated antibody conjugation Fig 3. Schematic illustration of streptavidin-biotin interaction-mediated antibody conjugation

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Absolute Mag™ Avidin Magnetic Particles
Absolute Mag™ Streptavidin Magnetic Particles

  • Covalent conjugation

Covalent antibody conjugation permanently immobilizes the antibody to the surface. Usually, antibodies’ primary amines (-NH2) are used and are chemically conjugated to functionalized particles. Aldehyde-, hydroxyl- and epoxy-activated particles are commonly used for these conjugations. The strong covalent binding between antibodies and particles prevents co-elution of antibodies along with the antigens. However, based on whichever antibodies’ amine group reacts with functionalized particles, the orientation of the coupled antibody is random. This random orientation may influence the antibody’s antigen-binding function and capacity, resulting in a declined antigen-capture efficacy.

 Schematic illustration of antibody covalent conjugation Fig 4. Schematic illustration of antibody covalent conjugation

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Absolute Mag™ Hydroxyl Magnetic Particles
Absolute Mag™ Epoxy Magnetic Particles
Absolute Mag™ Magnetic Particles Conjugation Kit

  • Hybrid conjugation

To overcome protein A/G’s low binding affinity to antibodies and covalent antibody conjugation’s random orientation issue, a hybrid conjugation strategy was developed. This hybrid conjugation takes the advantage of protein A/G’s specific binding affinity to antibodies’ Fc region and antibodies are immobilized on particles’ surface with their antigen-binding sites facing outward. Subsequently, chemical conjugations using crosslinkers strengthened the antibody-protein A/G binding by covalently link adjacent amines of the antibody and protein A/G. This hybrid conjugation strategy eliminates protein A/G-mediated antibody conjugation’s co-elution of antibody fragments and covalent conjugation’s random antibody orientation.

Schematic illustration of protein A/G-mediated antibody covalent conjugation Fig 5. Schematic illustration of protein A/G-mediated antibody covalent conjugation

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Immobilization of Antibody on Beads

Customized Protein Isolation (Streptavidin-biotin Interaction-mediated)

Apart from antibodies, many biomolecules can be recognized and bound by other molecules. For example, DNA binding proteins can be recognized and bound by DNA. Thus, these biomolecules can also be utilized for the isolation of their binding proteins.

Due to biotin’s strong binding affinity with avidin/streptavidin, avidin/streptavidin-biotin interaction has become a versatile and powerful strategy for all types of biomolecule conjugation. To isolate target proteins from mixtures using avidin/streptavidin-biotin interaction, biomolecules require biotin-labelling. Avidin/streptavidin-surface functionalized particles are used to extract biotinylated molecules from impurities. Diverse reagents or kits have been commercially available to assist researchers biotinylated their biomolecules of interest in their laboratories.

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Absolute Mag™ Avidin Magnetic Particles
Absolute Mag™ Streptavidin Magnetic Particles

Antibody Purification

The antibody is a crucial biomolecule in modern manufacturing and research. Traditional antibody purifications using ammonium sulfate precipitation or chromatography require a large quantity of crude samples for the process and these methods all face a big problem of product loss.

For researchers working in laboratories, isolating antibodies using protein A/G-conjugated particles is more common. Protein A/G-immobilized particles are able to capture antibodies through protein A/G’s binding to antibodies’ Fc region. Subsequently, when these antibody-immobilized particles are exposed to low-pH conditions, the antibody-protein A/G complex is disassembled, leading to the release of purified antibodies from particles.

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Absolute Mag™ Protein A Magnetic Particles
Absolute Mag™ Protein G Magnetic Particlest

Supplementary Reading

How to choose the correct particles size for protein separation
Various particles of different sizes can be chosen for protein or nucleic acid isolation. To decide which kind of particles should be used for the experiments, the separation time is the most important factor to be considered. Larger magnetic particles induce a larger magnetic field force, resulting in a shorter separation time. Whereas, due to the smaller magnetic field force, small magnetic beads offer a more mild way for protein separation. Magnetic beads with large size (1 µm) will become the best choice even if the magnetic beads with smaller size have higher binding capacity.

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