What Are Protein A Magnetic Beads? Application & Overview

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Protein A magnetic beads are an affinity matrix for the purification and isolation of antibodies or immunoglobulins. The beads are formed by covalently linking the truncated form of recombinant Protein A to a nonporous superparamagnetic particle.

Protein A is a cell surface protein found in the cell wall of Staphylococcus aureus. It’s 49 kDa in molecular weight and helps bacteria in their virulence and survival. Protein G is another protein that has many structural and functional similarities with protein A. Both proteins A/G have binding affinity for the mammalian immunoglobulin G (IgG) antibodies, such as human IgG.

Figure: Structure of the Staphylococcus aureus Protein A domain (left side) and binding of immunoglobulin Fc region with the minimized domain of protein A (right side).

One fine line of difference between protein A and G is that protein A has a high affinity for dog, cat, pig, and rabbit IgG, whereas protein G has a greater affinity for human and mouse IgG.

Protein A is often used in lab workflows coupled to a range of particles or molecules, such as biotin, enzyme, fluorescent dye, and radioactive iodine. One must-know point is that binding to these molecules does not affect its antibody binding site or its binding affinity.

Moreover, the protein is also coupled to latex, magnetic, and agarose beads to form affinity matrices for research use based on the goal of the study. A few examples of lab assays utilizing protein beads include immunoprecipitation, western blotting, or chromatography techniques.

In this article, we will cover how protein magnetic beads work, how they are utilized in lab workflows, and the industries that extensively exploit the tool for their biological research or bio-based product development.

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Properties of Protein A Magnetic Beads

Protein A magnetic beads are a high-throughput, quick, and easy magnetic separation technique for immunoglobulins. However, magnetic beads also bind to many other different ligands and facilitate their separation, such as nucleic acids (DNA and RNA or mRNA), plasmids, proteins, and peptides.

Given below is a characteristic feature of Protein A magnetic beads:

  • It is possible to regenerate the matrix without losing its binding capacity.
  • The bead has a high affinity for the immunoglobulin subclass of IgG from a range of species, such as mice, pigs, and rabbits. 
  • There is a stable and leak-resistant linkage between the protein and magnetic beads over a wide pH range. It allows the immunomagnetic purification of IgG from serum, ascites, or cell culture supernatants.
  • 1 ml of Protein A Magnetic Beads binds >280 g of Human IgG. 
  • In combination with a primary antibody, the Protein A bead can be used to immunoprecipitate target proteins from crude cell lysates. Additionally, reusable immunoprecipitation beads can be created by cross-linking specific antibodies to the Protein A surface. This can even prevent antibody co-elution with target antigens.
  • As opposed to traditional IP methods that rely solely on centrifugation, magnetic beads eliminate resin loss and provide more effective separation.
  • There is less nonspecific binding compared to agarose or Sepharose beads due to consistent IgG binding consistency.
  • They can either be used manually, such as with a magnetic stand, or with automated platforms.

Protocol to Use Protein A Magnetic Beads

Here’s a general protocol of purification involving Protein A magnetic beads. (NOTE: You need to tweak the protocol based on your research goal or based on the direction of use that comes with the commercial Protein A magnetic beads (mostly supplied in a 20% slurry). Go through the safety data sheets (SDS) carefully.

Before starting a protocol dilute the required amount of antibody in the binding buffer to prepare an antibody solution. The samples used in the protocol can be separated by using SDS-PAGE electrophoresis. The purification procedure is consist of six stages that are as follows:

  1. Magnetic bead preparation
  • Dispense magnetic beads into a 1.5 ml microfuge tube as required.
  • Remove the storage solution (consisting of sodium azide, PBS, BSA, and Tween 20) and place the tube in the magnetic rack.
  • Add a 500 µl binding buffer.
  • Resuspend the beads.
  • Remove the liquid 
  1. Antibody capture
  • Add the antibody solution immediately
  • Resuspend and mix for at least 15 minutes
  • Remove the liquid
  1. Washing (repeat twice)
  • Add 500 µl Binding Buffer containing 0.5 M NaCl
  • Remove the liquid
  1. Target binding
  • Add sample diluted in binding buffer
  • For 60 minutes, incubate with slow end-over-end mixing
  • Remove and collect the unbound fraction
  1. Washing
  • Add 500 µl Binding Buffer containing 0.5 M NaCl
  • Remove the liquid
  1. Elution
  • Add 2 volumes of elution buffer
  • For a minimum of two minutes, resuspend and incubate the beads
  • Remove and collect the elution fraction

The same steps and reagents can be followed with protein A/G magnetic beads. However, the output and yield won’t be equal.

Figure: An illustrative diagram of the antibody purification steps.

What Are Protein A Magnetic Beads Used For?

The ease of the use of protein A magnetic bead makes them suited for a range of lab applications such as:

  • Co-immunoprecipitation (Co-IP)
  • Chromatin immunoprecipitation
  • Antibody and protein purification
  • Sample preparation for next-generation sequencing (NGS) and PCR
  • molecular and immunodiagnostics

Immunoprecipitation assay

It’s a technique for small-scale purification of antigens using a specific antibody immobilized in a magnetic bead matrix. The protein A has a high affinity for the heavy chains of the antibody Fc region. This immobilizes the antibody to the solid support. When the sample is added to the matrix, the antigen binds to the antibody. In the last step, the complex is eluted using an elution buffer.

Figure: An illustration of the immunoprecipitation steps.

What Industries Use Protein A Magnetic Beads?  

Protein A magnetic beads are used in a variety of Life Sciences labs and industries for research use, biopharmaceutical production, and disease diagnostic processes. 

Biopharmaceutical

Protein A has a high affinity for IgG. Thus, Protein A magnetic beads are widely used for the purification of IgG immunoglobulins. The monoclonal IgG antibodies have therapeutic applications in many life-threatening diseases, such as cancer and autoimmune disorders.

Biotechnology

Protein A magnetic antibodies are extensively exploited in biotech labs in a spectrum of applications. It has uses in protein and antibody purification, diagnostic procedures, immunoprecipitation, cell isolation, and IgG antibody enrichment.  

Figure: Steps to antibody purification using Protein A magnetic beads.

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Protein A magnetic beads are high-affinity matrices made of Protein A coupled to superparamagnetic particles. It has applications in a range of high-throughput lab assays, such as immunoprecipitation, western blotting, and antibody and protein purification. In biopharmaceutical labs, the protein A beads are also used in therapeutic procedures for many diseases, such as cancers.

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