Immunoprecipitation products and solutions

Products and solutions for immunoprecipitation (IP)

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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 are still one of the best approaches.

IP starter-packs

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Benefits of magnetic beads for IP
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.

Summary of benefits for Dynabeads technology:

How it works (IP with magnetic beads)
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 remove of unbound antibodies.
  2. Add sample to the beads. Add your protein-containing sample to the washed beads with bound antibody 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 is sufficient.
  3. Wash the protein-bound beads. To ensure high purity of the bound target protein, the beads should be washed 2–4 times with buffer on the magnet to remove all unbound proteins. The efficient isolation process ensures a high signal-to-noise ratio.
  4. Elute the protein.
Process and Benefits of IP with Magnetic Dynabeads

Protein elution properties

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 the downstream assay.

Antibody-binding product selection guide

Antibody-binding product selection guide

 Protein A, G, A/G
Surface-activated beads (epoxy)*
Binding propertiesNon-covalent antibody bindingNon-covalent antibody bindingCovalent antibody binding
Antibody co-eluted off the beadsSim.Sim.Não
Tipo de comunicaçãoAnti-mouse binds mouse IgG1, IgG2a, IgG2b; anti-rabbit binds all rabbit IgGsAll antibodies
Mass spec compatible
NãoSim
Nonspecific bindingBaixoBaixoUltra-low
Antibody coupling time & temperature (RT=room temp.)
  • Proteína A 10 min, RT
  • Protein G: 10–40 min, RT
  • Protein A/G: 60 min, RT
>30 min, 2–8°C16–24 hr, 37°C
Products

CD apenas

Kits:

CD apenas

Kits:


† Crosslinking can be performed to avoid co-elution of the antibody, but this can decrease the yield of the target antigen.

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 include:

  • 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. 

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 for Streptavidin Dynabeads is for purification of nucleic acids and next generation sequencing (NGS) workflows, but they are also used for immunoprecipitation (IP).

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

Popular fusion tags for recombinant protein expression include:

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.

Antibody-binding IP products

Choose this if you have an antibody that recognizes your protein

Your choice of antibody binding products depends on the downstream assay.

Antibody-binding product selection guide

Antibody-binding product selection guide

 Protein A, G, A/G
Surface-activated beads (epoxy)*
Binding propertiesNon-covalent antibody bindingNon-covalent antibody bindingCovalent antibody binding
Antibody co-eluted off the beadsSim.Sim.Não
Tipo de comunicaçãoAnti-mouse binds mouse IgG1, IgG2a, IgG2b; anti-rabbit binds all rabbit IgGsAll antibodies
Mass spec compatible
NãoSim
Nonspecific bindingBaixoBaixoUltra-low
Antibody coupling time & temperature (RT=room temp.)
  • Proteína A 10 min, RT
  • Protein G: 10–40 min, RT
  • Protein A/G: 60 min, RT
>30 min, 2–8°C16–24 hr, 37°C
Products

CD apenas

Kits:

CD apenas

Kits:


† Crosslinking can be performed to avoid co-elution of the antibody, but this can decrease the yield of the target antigen.
Biotin-binding IP products

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 include:

  • 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. 

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 for Streptavidin Dynabeads is for purification of nucleic acids and next generation sequencing (NGS) workflows, but they are also used for immunoprecipitation (IP).

Recombinant protein (fusion tag) IP products

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

Popular fusion tags for recombinant protein expression include:

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.

Superior:

Benchmarking data: Dynabeads vs. other IP solutions

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.

Dynabeads compared to resin-based solutions

Figura 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

Figura 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.

Figura 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.

IP Starter Packs – Get a magnet and Dynabeads together and save 21 to 24%

Each starter pack has the DynaMag-2 magnet. Choose the starter pack best suited for you.
These bundled combinations save you between 21–24% compared to purchasing separately.

Magnet with Dynabeads only
Número de CatContém Número de dispensadores
10013DDynabeads Protein A, 2 x 1 mL + DynaMag-240
10014DDynabeads Protein G, 2 x 1 mL + DynaMag-240
10015D
40
10016DDynabeads Protein A, 5 mL + DynaMag-2100
10017DDynabeads Protein G, 5 mL + DynaMag-2100
Magnet with Dynabeads IP Kit
Número de CatContém Número de dispensadores
10018D
40
10019D
40
Immunoprecipitation publication trends
Immunoprecipitation publication trends

The publication trend for immunoprecipitation says it all. The number of publications citing the use of Dynabeads magnetic beads vs. other technologies and products for immunoprecipitation procedures is skyrocketing.

Dynabeads IP citations

Dynabeads IP citations.

Dynabeads chIP citations

Dynabeads ChIP citations.

ChIP, RIP, and Protein-Nucleic Acid Pull-Down Products

Automated immunoprecipitation with downstream Mass Spec analysis

KingFisher Flex

Following immunoprecipitation with Dynabeads magnetic beads, the typical downstream analysis of protein is performed by Western blot or Mass spectrometry. The growing number of researchers process multiple samples at a time, and automate the IP process on KingFisher instruments. Below are several recent publications describing some of the typical workflows for IP with Dynabeads on KingFisher Flex platform, followed by Mass spec analysis.

Vídeos
Recursos

For Research Use Only. Not for use in diagnostic procedures.