Products and solutions for immunoprecipitation (IP)

Publication trends show that the fastest growing method for immunoprecipitation incorporates the use of magnetic beads and that Invitrogen™ Dynabeads™ products are the most used and referenced products in this trend. Why? Because Dynabeads magnetic beads offer you the best balance of capacity/yield, reproducibility, purity, and cost for smaller-scale isolation of specific proteins (e.g., immunoprecipitation, IP) and protein complexes (co-immunoprecipitation, co-IP).

Protein purification and research needs have changed considerably since the introduction of the agarose-based Sepharose™ slurry in the 1970’s when the focus was purifying large amounts of protein or antibody. If this is your primary application, agarose/resin-based columns still work well. 


Benefits of magnetic beads for IP

Magnetic beads have become the gold standard for immunoprecipitation and pull-down assays because they offer a faster, easier, and more efficient way of pulling down proteins of interest than traditional Sepharose™ agarose or other agarose-based resins. Dynabeads magnetic beads help ensure that you achieve the best balance of high yield and reproducibility with low nonspecific binding and low cost. Check out these IP Myth videos that provide a great way to visually understand the benefits of Dynabeads magnetic beads.

Summary of benefits for Dynabeads technology:

  • Low background—little to no nonspecific binding and no preclearing required for high-purity protein yields.
  • Highly reproducible—uniform beads ensure the most consistent results.
  • Highly sensitive—most-cited method for sensitive applications, such as ChIP and IP, of low- abundance proteins.
  • Fast and easy— <40 min protocol with no centrifugation or preclearing steps.
  • Antibody savings—all binding occurs on the smooth outer surface of the beads, which conserves precious antibodies and supports a cost-efficient solution per sample.
  • Flexible—products for IP, Co-IP, pull-down, and ChIP assays; ideal for manual and automated protocols.
  • Automation of IP protocol—learn how the Thermo Scientific™ KingFisher™ platform now supports immunoprecipitation automation.
How it works (IP with magnetic beads)

Immunoprecipitation is easy and fast when using Dynabeads magnetic beads as a solid support for protein isolation and can be done in four simple steps:

  1. Binding of the antibody to the beads. Dynabeads magnetic beads are precoated with protein A, protein G, anti-mouse IgG, or anti-rabbit IgG antibodies and due to the rapid kinetics will bind the added antibody in only 10 minutes. Biotinylated antibodies can also be used in combination with streptavidin-coupled Dynabeads. Wash the beads once on the magnet to get rid of unbound antibodies.
  2. Add sample to the beads. Add your protein-containing sample to the washed, antibody-bound beads and incubate for another 10 minutes to bind the target protein of interest. If the protein is of low abundance or low affinity, the incubation can be increased to 1 hour or an overnight incubation; but in general, a 10-minute incubation period is sufficient.
  3. Wash the protein-bound beads. To secure bound target protein with high purity, the beads should be washed 2–4 times with buffer on the magnet to remove all unbound proteins. The efficient isolation process secures a high signal-to-noise ratio.
  4. Elute the protein. If required, the protein can be eluted off the beads by using either a mild elution procedure or a denaturing elution procedure (for details, see elution description below).

This procedure is simple, fast, requires no preclearing step, and results in high-target protein purity, yield, and consistent results.
 

Protein elution properties

The recombinant protein A and/or protein G on the beads contains no albumin binding sites, thus avoiding co-purification of contaminating proteins. Binding of antibodies to the beads in solution is an equilibrium reaction. To capture as many antibodies as possible, the reaction volume should be kept low to maintain high concentrations of beads and antibody. There is no need for pre-treating the sample (even viscous samples), hence no dilution of sample or beads should be done. If the antibody concentration in the sample is suspected to be very low, the amount of beads can be increased.

As an alternative to eluting antibody from the beads, the Dynabeads may be re-suspended in a Na-phosphate buffer. To prevent co-elution, it is possible to cross-link the antibody to protein A/G on the beads prior to immunoprecipitation. This is not necessary for downstream SDS-PAGE followed by autoradiography or western blotting, and optional for silver or coomassie dye staining.

Find the right immunoprecipitation product for your experiment

Choose this if you have an antibody that recognizes your protein

Your choice of antibody binding products depends on whether your downstream assay is mass spectrometry, or if you don’t want the antibody co-eluted with your target protein, etc.

Antibody binding is the most common method and is used when your target antibody can be bound directly to the beads or indirectly to a pre-coated ligand on the magnetic beads.

Antibody-binding product selection guide

  Protein A, G, A/G Secondary antibodies
(anti-mouse, -rabbit)
Surface- activated beads (epoxy)*
Binding properties Non-covalent antibody binding Non-covalent antibody binding Covalent antibody binding
Antibody co-eluted off the beads Yes Yes No
Type of ligand Different ligands bind different species and antibody subclasses with different specificity Anti-mouse binds mouse IgG1, IgG2a, IgG2b; anti-rabbit binds all rabbit IgGs All antibodies
Mass spec compatible Yes (Protein A/G);
Not validated (Protein A, G)
No Yes
Non-specific binding Low Low Ultra-low
Antibody coupling time & temperature (RT=room temp.)
  • Protein A: 10 min, RT
  • Protein G: 10–40 min, RT
  • Protein A/G: 60 min, RT
>30 min, 2–8°C 16–24 hr, 37°C
Products

Beads only:

Kits:

Beads only:

Kits:

* See more choices in surface-activated Dynabeads for the binding and capture of additional targets.
† Crosslinking can be performed to avoid co-elution of the antibody, but this can decrease the yield of the target antigen.
‡ Learn which Antibody-binding Proteins are best for your IP antibody.

Choose this if you have a biotinylated antibody (or ligand) that recognizes your protein

Main advantages for using a biotinylated antibody with streptavidin-coated beads for IP

  • If you have a sample rich in soluble IgGs
  • If you have a recombinant antibody lacking Fc regions
  • If you already have streptavidin-coated Dynabeads in the lab
  • If you need a bead compatible with mass spectrometry (secondary-coated and epoxy-coated Dynabeads are also compatible with mass spectrometry)

To allow flexibility, four different types of streptavidin-coupled Dynabeads are available. View the Dynabeads streptavidin selection guide to see the different characteristics.

Over the past 25 years, streptavidin-coupled Dynabeads in combination with a biotinylated ligand have been widely used and are cited in thousands of papers for diverse manual and automated applications. The most widely used application is for purification of nucleic acids (NA), but it is also used for immunoprecipitation (IP).

Choose this if you have a recombinant (fusion tagged) protein

Popular fusion tags for recombinant protein expression

  • His tag—consists of a string of six to nine histidine residues; primarily used for purification via immobilized metal affinity chromatography (IMAC)
  • GST tag—consists of glutathione S-transferase (GST), a complete 211 amino acid protein (26 kDa); primarily used for purification via glutathione agarose resin
  • HA tag—consists of a peptide (YPYDVPDYA) derived from the human influenza hemagglutinin (HA) protein; immobilized anti-HA antibodies are used to immunoprecipitate HA-tagged recombinant proteins
  • c-Myc tag—consists of a peptide (EQKLISEEDL) derived from the human c-myc oncogene (p62 c-myc); immobilized anti-c-Myc antibodies are used to immunoprecipitate c-Myc-tagged recombinant proteins. 

His and GST are not true epitope tags, because they are not usually purified or detected via specific antibodies. By contrast, HA and c-Myc tags are true epitope tags, because their only means of purification or detection is via specific antibodies. Epitope tags are seldom used for bulk purification but are most often used for immunoprecipitation or co-immunoprecipitation (IP, co-IP). Nevertheless, all four of these tag systems can be used for either purification or pull-down applications.

If you’ve got a biotinylated antibody that recognizes the protein you wish to capture, you can create your own affinity product with one of these products.

Benchmarking data: Dynabeads vs. other IP solutions

This section includes comparative data showing results of Dynabeads versus other magnetic solutions as well as resin-based solutions. If you are still using Sepharose or agarose bead slurry for IP, you may also be interested in seeing these informative animations debunking 4 Myths about Immunoprecipitation.

Dynabeads compared to resin-based solutions

Figure 1. Shorter protocol time and better yields with Dynabeads magnetic beads. Equal amounts of sample material with respect to Ab content and cell lysate volume were used for all IP protocols according to the manufacturer’s protocol. For the Dynabeads-based method, all the antibodies on the bead surface are accessible for optimal, highly reproducible antigen binding.

Dynabeads compared to magnetic solutions

Figure 2. The magnetic bead you choose will affect your results. Dynabeads magnetic beads have a defined surface to carry out the necessary binding, with no inner surface to trap any unwanted proteins. (A) Dynabeads products are the most uniform, monodisperse superparamagnetic beads, manufactured with highly controlled product qualities to enable a high degree of reproducibility. (B–D) Magnetic particles from alternative suppliers have variable shapes and sizes that trap impurities, resulting in lower reproducibility and increased nonspecific binding.

Figure 3. Benchmarking data shows that Dynabeads magnetic products have the best performance when taking into account the combination of highest yield and lowest nonspecific binding. Our magnetic beads also have the fastest protocol, helping improve your lab life by removing unnecessary steps.