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Immunoprecipitation with Magnetic Beads |
Dynabeads and Pierce magnetic beads offer 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). Utilization of Dynabeads magnetic beads is continually shown to be the fastest growing method for IP in publication trends around the world.
Dynabeads
- 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 to perform immunoprecipitation using Dynabeads and Pierce magnetic beads
Step 1*: Binding of the antibody to the beads
Binding of the antibody to the beads. Dynabeads and Pierce 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 and Pierce magnetic beads. Wash the beads once on the magnet to remove of unbound antibodies.
Step 2*: Add sample to the beads
Add sample to the beads. Add the 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.
Step 3: Wash the protein-bound beads
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.
Step 4: Elute the protein
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).
*Pierce magnetic beads can perform same steps with slightly longer protocol time to complete.
Click image to enlargeThis procedure is simple, fast, requires no preclearing step, and results in high-target protein purity, yield, and consistent results.
Use Thermo Scientific Magnetic Beads for your IP. Animated representation of the IP process with magnetic beads.
Varying degrees of elution with Dynabeads or Pierce Magnetic Beads
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.
Use the Thermo Scientific Pierce Crosslink Magnetic IP/Co-IP Kit as an alternative to eluting antibody from the beads. 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 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, anti-rabbit) | Surface-activated beads | |
|---|---|---|---|
| Products | Beads only:Kits: | Beads only: | Beads only:Kits: |
| 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 (Pierce MS-Compatible Magnetic IP Kit, protein A/G) Not validated (Protein A, G) | No | Yes |
| Nonspecific binding | Low | Low | Ultra-low |
* 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 of magnetic beads:
- If you have a sample rich in soluble IgGs
- If you have a recombinant antibody lacking Fc regions
- If you need a bead 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 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 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.
- FLAG tag—consists of a peptide (DYKDDDDK) which is recognized by an immobilized high-affinity rat monoclonal antibody (clone L5); primarily used for the isolation of protein complexes with multiple subunits, because the mild purification process tends not to disrupt these interactions
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 IP or co-IP. Nevertheless, all four of these tag systems can be used for either purification or pull-down applications.
Benchmarking data: Dynabeads vs. other IP solutions
Dynabeads continually outperform other methods and magnetic bead solutions for IP. Still using Sepharose or agarose bead slurry? See some of the most common myths about immunoprecipitation.
Dynabeads compared to resin-based solutions
Click image to enlarge
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
Click image to enlarge
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.
Click image to enlarge
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.
Automate immunoprecipitation with KingFisher instruments
Perform direct or indirect immunoprecipitation using Dynabeads and Pierce magnetic beads on KingFisher Instruments.
Dynabeads
- 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 to perform immunoprecipitation using Dynabeads and Pierce magnetic beads
Step 1*: Binding of the antibody to the beads
Binding of the antibody to the beads. Dynabeads and Pierce 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 and Pierce magnetic beads. Wash the beads once on the magnet to remove of unbound antibodies.
Step 2*: Add sample to the beads
Add sample to the beads. Add the 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.
Step 3: Wash the protein-bound beads
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.
Step 4: Elute the protein
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).
*Pierce magnetic beads can perform same steps with slightly longer protocol time to complete.
Click image to enlargeThis procedure is simple, fast, requires no preclearing step, and results in high-target protein purity, yield, and consistent results.
Use Thermo Scientific Magnetic Beads for your IP. Animated representation of the IP process with magnetic beads.
Varying degrees of elution with Dynabeads or Pierce Magnetic Beads
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.
Use the Thermo Scientific Pierce Crosslink Magnetic IP/Co-IP Kit as an alternative to eluting antibody from the beads. 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 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, anti-rabbit) | Surface-activated beads | |
|---|---|---|---|
| Products | Beads only:Kits: | Beads only: | Beads only:Kits: |
| 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 (Pierce MS-Compatible Magnetic IP Kit, protein A/G) Not validated (Protein A, G) | No | Yes |
| Nonspecific binding | Low | Low | Ultra-low |
* 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 of magnetic beads:
- If you have a sample rich in soluble IgGs
- If you have a recombinant antibody lacking Fc regions
- If you need a bead 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 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 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.
- FLAG tag—consists of a peptide (DYKDDDDK) which is recognized by an immobilized high-affinity rat monoclonal antibody (clone L5); primarily used for the isolation of protein complexes with multiple subunits, because the mild purification process tends not to disrupt these interactions
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 IP or co-IP. Nevertheless, all four of these tag systems can be used for either purification or pull-down applications.
Benchmarking data: Dynabeads vs. other IP solutions
Dynabeads continually outperform other methods and magnetic bead solutions for IP. Still using Sepharose or agarose bead slurry? See some of the most common myths about immunoprecipitation.
Dynabeads compared to resin-based solutions
Click image to enlarge
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
Click image to enlarge
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.
Click image to enlarge
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.
Automate immunoprecipitation with KingFisher instruments
Perform direct or indirect immunoprecipitation using Dynabeads and Pierce magnetic beads on KingFisher Instruments.
Ordering information
Publications
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 ChIP citations.
- Brinkmalm A, Öhrfelt A, Bhattacharjee P, Zetterberg H. Detection of α-Synuclein in Biological Samples Using Mass Spectrometry.Methods Mol Biol. 2019;1948:209-220. doi: 10.1007/978-1-4939-9124-2_16.
- Cicognola C, Brinkmalm G, Wahlgren J, Portelius E, Gobom J, Cullen NC, Hansson O, Parnetti L, Constantinescu R, Wildsmith K, Chen HH, Beach TG, Lashley T, Zetterberg H, Blennow K, Höglund K. Novel tau fragments in cerebrospinal fluid: relation to tangle pathology and cognitive decline in Alzheimer's disease.Acta Neuropathol. 2019 Feb;137(2):279-296. doi: 10.1007/s00401-018-1948-2. Epub 2018 Dec 13.
- Gkanatsiou E, Portelius E, Toomey CE, Blennow K, Zetterberg H, Lashley T, Brinkmalm G. A distinct brain beta amyloid signature in cerebral amyloid angiopathy compared to Alzheimer's disease. Neurosci Lett. 2019 May 14;701:125-131. doi: 10.1016/j.neulet.2019.02.033. Epub 2019 Feb 23.
- Kvartsberg H, Lashley T, Murray CE, Brinkmalm G, Cullen NC, Höglund K, Zetterberg H, Blennow K, Portelius E. The intact postsynaptic protein neurogranin is reduced in brain tissue from patients with familial and sporadic Alzheimer's disease. Acta Neuropathol. 2019 Jan;137(1):89-102. doi: 10.1007/s00401-018-1910-3. Epub 2018 Sep 22.
- Öhrfelt A, Brinkmalm A, Dumurgier J, Brinkmalm G, Hansson O, Zetterberg H, Bouaziz-Amar E, Hugon J, Paquet C, Blennow K. The pre-synaptic vesicle protein synaptotagmin is a novel biomarker for Alzheimer's disease.Alzheimers Res Ther. 2016 Oct 3;8(1):41.
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