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Magnetic Beads for Immunoprecipitation |
Immunoprecipitation (IP) is a key technique in cellular and molecular biology, employed to isolate or purify specific antigens (proteins of interest) using solid phases like agarose or Sepharose™ resin, or magnetic particles. The IP method was initially performed on small aliquots of agarose resin and is used when scale-up is anticipated. Magnetic beads are versatile, enabling the targeting of single proteins or peptides (IP), studying protein interactions through protein complexes (Co-IP), identify regions of the genome with DNA-binding or RNA-binding proteins (ChIP or RIP), and exploring the functional dynamics of protein-nucleic acid interactions.
On this page:
- Advantages of magnetic beads for immunoprecipitation
- Immunoprecipitation beads selection guides
- Microplastic-free magnetic beads for IP
- Automated immunoprecipitation protocols
- What is immunoprecipitation?
- Videos on immunoprecipitation
- Immunoprecipitation FAQs
- Immunoprecipitation beads publications
- Ordering information
- Dynabeads selection guide
- Resources for immunoprecipitation
Advantages of magnetic beads for immunoprecipitation
Magnetic beads for IP have become more common, especially compared to the traditional method of using agarose slurries or resin for protein isolation. This is because magnetic immunoprecipitation beads provide several advantages over the traditional method, such as:
- Low background—Little to no non-specific binding and no preclearing required for high-purity protein yields
- Highly reproducible—Uniform composition of the magnetic beads helps ensure the most consistent results
- Highly sensitive—Most-cited method for sensitive applications, such as ChIP and IP for 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 compared to porous agarose beads; using beads for IP is a cost-efficient solution per sample
- Automation compatible—Immunoprecipitation beads protocol can be automated on the Thermo Fisher Scientific KingFisher platform
View common IP myths on agarose resins vs magnetic beads
Learn about co-immunoprecipitation
Immunoprecipitation beads selection guides
- Antibody-binding magnetic beads—Most common method
- Fusion tag-binding magnetic beads—If your protein is a recombinant protein
- Biotin-binding magnetic beads—If your protein is biotinylated
- Surface-activated magnetic beads—If your ligand or target sample is tricky
Antibody-binding magnetic beads for IP
| Protein A, G, A/G | Secondary antibodies (anti-mouse, anti-rabbit) | |
|---|---|---|
| Recommended products | Beads only:Kits: | Beads only: |
| Binding properties | Non-covalent antibody binding | |
| Antibody co-eluted off the beads | Yes* No (Pierce Crosslink Magnetic IP/Co-IP Kit) | Yes* |
| Type of ligand | Different ligands bind different species and antibody subclasses with different specificity† |
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| Non-specific binding | Low | |
* 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.
Learn more about how mass spectrometry and western workflows are enhanced by high-performance protein enrichment using Dynabeads and DynaGreen magnetic beads in our magnetic bead product brochure
Dynabeads magnetic beads compared to Sepharose- and agarose-based solutions
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Figure 1. Shorter protocol time and better target protein 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 magnetic beads compared to competitor magnetic beads
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Figure 2. Electron microscopy of Dynabeads magnetic beads compared to competitor magnetic beads. Dynabeads and DynaGreen magnetic beads have a defined surface to carry out the necessary binding, with no inner surface to trap any unwanted proteins. Dynabeads products (A) are the most uniform, monodisperse superparamagnetic beads, manufactured with highly controlled product qualities to help ensure the highest degree of purity and reproducibility, while the DynaGreen products (B) offer high purity and higher binding capacity vs. the Dynabeads products, but also offer a more sustainability-conscious version of Dynabeads products. Magnetic particles from alternative suppliers (C–E) have variable shapes and sizes that trap impurities, resulting in lower purity and reproducibility without proper functionalization and antibody coupling.
Dynabeads magnetic beads compared to competitor magnetic beads for IP
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Figure 3. Dynabeads magnetic beads have exceptional immunoprecipitation performance with the combination of highest yield and lowest non-specific binding. (A) Silver staining of the protein membrane shows that Dynabeads magnetic beads have the most enriched binding at the antibody main band and very low non-specific binding with low signal/noise. (B) Western blot analysis of CD81 pulldown in Jurkat cells show that Dynabeads Protein G beads have the highest yield with a short ten-minute protein incubation.
Fusion tag-binding magnetic beads for IP
| Tag | c-Myc | FLAG | HA | Histidine | GST |
|---|---|---|---|---|---|
| Description |
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Recommended products |
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.
Biotin-binding magnetic beads for IP
Main advantages of magnetic beads for biotin-binding:
| Recommended products | Dynabeads M-280 Streptavidin | Dynabeads MyOne Streptavidin T1* | Pierce Streptavidin Magnetic Beads |
|---|---|---|---|
| Description |
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| Capacity |
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* Low sedimentation rate and improved reaction kinetics compared to M-280 beads.
Surface-activated magnetic beads for IP
If your target protein or ligand needs to be covalently coupled, use surface-activated beads
Different ligands (antibodies, proteins, etc.) require different bead surface properties and immobilization chemistries. Choose surface-activated beads to control factors such as:
- Hydrophobicity: choose hydrophilic of hydrophobic beads
- Bead size: 1 µm, 2.8 µm, and 4.5 µm beads available
- Surface chemistry: epoxy, amine, or tosylactivated
Searching for high-performing, sustainable magnetic beads? Introducing DynaGreen Magnetic Beads.
DynaGreen Magnetic Beads provide high-quality, reproducible results and are also environmentally friendly and microplastic-free. How do these beads contribute to a more sustainable science? All DynaGreen Magnetic Beads have the following features:
- Non-microplastic bead core
- Energy-efficient manufacturing
- Reduced water consumption
- Use of non-hazardous chemicals
- Recyclable packaging
- Paperless documents
Detect and analyze proteins in as little as 40 minutes
With automation protocols available for KingFisher instruments, you can be ready to detect and analyze proteins in as little as 40 minutes with minimal hands-on time.
What is immunoprecipitation?
Immunoprecipitation (IP) is a cellular and molecular biology method used in laboratories to isolate or purify a specific antigen (or protein of interest) via a solid phase (agarose or Sepharose™ resin or magnetic particles). The IP method was first performed on small aliquots of agarose resin in microcentrifuge tubes. The IP method has evolved to use superparamagnetic particles to specifically target proteins of interest.
How does immunoprecipitation work?
The Dynabeads magnetic beads IP procedure is simple, fast, requires no preclearing step, and results in high-target protein purity, yield, and consistent results. Both DynaGreen magnetic beads and Pierce magnetic beads can perform the same steps with slightly longer protocol times to complete. DynaGreen protocol finishes in <80 min. Pierce protocol finishes in <120 min.
Step 1: Binding of the antibody to the beads
Dynabeads, DynaGreen, and Pierce magnetic beads come pre-coated with protein A, protein G, anti-mouse IgG, or anti-rabbit IgG antibodies. Their rapid kinetics allow them to bind to the added antibody in about ten minutes (Figure 4). Biotinylated antibodies can also be used in combination with streptavidin-coupled Dynabeads and Pierce magnetic beads. After antibody incubation a wash step is performed to remove unbound antibodies.
Step 2: Add sample to the beads
The protein-containing sample is added to the beads-antibody mix and incubated for ten minutes to allow target protein binding. If the protein is of low abundance or low affinity, the incubation time can be increased to one hour or overnight. For most cases, a ten-minute incubation is sufficient.
Step 3: Wash the protein-bound beads
To help ensure high purity of the bound target protein, the beads should be washed 2 to 4 times with buffer on the magnet to remove all unbound proteins. This step in the isolation process helps ensure a high signal-to-noise ratio.
Step 4: Elute the protein
The protein can be eluted off the beads by using either a mild elution procedure or a denaturing elution procedure (Figure 5).
Click image to enlarge
Figure 4. Immunoprecipitation protocol with magnetic beads.
Figure 5. Types of elution buffers for immunoprecipitation with magnetic beads. Depending on your target protein and how they bind to the magnetic beads, there are three main types of elution buffers to unbind the target protein. Denaturing elution buffers are used for SDS-PAGE. Mild elution buffers are used for protein characterization, immunization, enzyme studies, amino acid sequence determination, and crystallization. Protein-protein interactions, enzyme studies, bioassays, and immunoassays may be performed without eluting your target protein from the magnetic beads.
Videos on immunoprecipitation
Covalent Coupling of Antibodies to Magnetic Beads - Fast and EfficientThe benefits of the Dynabeads Antibody CoupliKit include: • Use any primary antibody • Get ultra-low non-specific binding • No need for cross-linking since Antibody is irreversibly bound to the beads and will not be eluted off with target protein
IP Myth 4 Dynabeads are expensiveIP Myth 4 Dynabeads are expensive
IP Myth 3 Capacity is crucialIP Myth 3 Capacity is crucial
IP Myth 2 Pre-clearing is necessaryIP Myth 2 Pre-clearing is necessary
IP Myth 1 Background can't be avoidedIP Myth 1 Background can't be avoided
How To Use Dynabeads For ImmunoprecipitationHow to use Dynabeads for immunoprecipitation
Automated Immunoprecipitation in 40 min using Dynabeads and KingFisher FlexFor IP, KingFisher instruments with Dynabeads offer the best balance of high yield and reproducibility with low nonspecific binding and cost, which is one reason why they have become the gold standard for immunoprecipitation using magnetic beads.
Immunoprecipitation Publication Trends - The Reasons for the ShiftImmunoprecipitation Publication Trends - The Reasons for the Shift
Immunoprecipitation with Dynabeads magnetic beads FAQs
Here are some frequently asked questions regarding the use of Dynabeads magnetic beads for immunoprecipitation.
A: Dynabeads Protein A and Dynabeads Protein G are 2.8-micron beads covalently coupled with Protein A and recombinant Protein G, respectively. Protein A and Protein G differ in their binding strength to immunoglobulins from different species and subclasses.
A: 1. Verify binding/specificity of your antibody to your antigen (e.g., by ELISA).
2. Check the binding of your antibodies to the beads. If the antibodies are not captured and bound to the beads, the immunoprecipitation experiment will not work.
3. If you have used the indirect method of immunoprecipitation, try the direct method. Conversely, if you have used the direct method, try the indirect method.
4. Check the amount of beads and sample volume. With reference to the capacity of different beads proposed in the package inserts, increase the amount of beads or the concentration of your antibody during coupling.
5. Increase the incubation time.
6. Try another antibody.
A: Yes, you can use the larger beads. Note: Smaller beads provide a larger surface area and therefore give higher yields of protein compared to the larger 4.5-micron beads.
Immunoprecipitation publications using Dynabeads magnetic beads
- Brinkmalm A., Öhrfelt A., Bhattacharjee P., et al. (2019) Detection of α-Synuclein in Biological Samples Using Mass Spectrometry. Methods Mol Biol 1948:209–220.
- Bafunno V., Firinu D., D’Apolito M., et al. (2018) Mutation of the angiopoietin-1 gene (ANGPT1) associates with a new type of hereditary angioedema. J Allergy Clin Immunol 141(3):1009–1017.
- Stanton BZ., Hodges C., Crabtree GR., et al. (2017) A General Non-Radioactive ATPase Assay for Chromatin Remodeling Complexes. Curr Protoc Chem Biol 9(1):1–10.
- Yang, Yuening et al. (2024) “Current progress in strategies to profile transcriptomic m6A modifications.” Frontiers in cell and developmental biology vol. 12 1392159.
- Liu, Cheng et al. (2023) “Rif1 Regulates Self-Renewal and Impedes Mesendodermal Differentiation of Mouse Embryonic Stem Cells.” Stem cell reviews and reports vol. 19,5: 1540-1553.
- Wagner, Susan et al. (2022) “Selective footprinting of 40S and 80S ribosome subpopulations (Sel-TCP-seq) to study translation and its control.” Nature protocols vol. 17,10: 2139-2187.
- Ben Maamar, Millissia et al. (2021) “Genome-Wide Mapping of DNA Methylation 5mC by Methylated DNA Immunoprecipitation (MeDIP)-Sequencing.” Methods in molecular biology vol. 2198: 301-310.
- Hsu, Hang-Kai et al. (2020) “Detection of DNA Methylation by MeDIP and MBDCap Assays: An Overview of Techniques.” Methods in molecular biology vol. 2102): 225-234.
View publications on cell isolation using Dynabeads products
View publications on Dynabeads Streptavidin magnetic beads
Additional publications can be found in our Citations and References database. The database can be searched using a product catalog number or keyword. For example, searching with the term "10001D" will return a list of journal articles that cite the use of Dynabeads Protein A for Immunoprecipitation (Cat. No. 10001D) and searching with the term "dynabeads" will return a list of journal articles that cite the use of any Dynabeads product. Note that the Citations and References database on thermofisher.com contains only the small subset of scientific journal articles that cite the use of our products. Many more can be found via targeted searches of the PubMed™ database and similar databases.
- Duan, Wan-Li et al. (2024) “N6022 attenuates cerebral ischemia/reperfusion injury-induced microglia ferroptosis by promoting Nrf2 nuclear translocation and inhibiting the GSNOR/GSTP1 axis.” European journal of pharmacology vol. 972.
- Xu, Jihao et al. (2024) “Engrailed-1 Promotes Pancreatic Cancer Metastasis.” Advanced science vol. 11,6.
- Murayama, Takahiko et al. (2024) “Targeting DHX9 Triggers Tumor-Intrinsic Interferon Response and Replication Stress in Small Cell Lung Cancer.” Cancer discovery vol. 14,3 468-491.
- Masato, Anna et al. (2024) “Sequestosome-1 (SQSTM1/p62) as a target in dopamine catabolite-mediated cellular dyshomeostasis.” Cell death & disease vol. 15,6 424.
- Li, Sumei et al. (2021) “Spastin Interacts with CRMP2 to Regulate Neurite Outgrowth by Controlling Microtubule Dynamics through Phosphorylation Modifications.” CNS & neurological disorders drug targets vol. 20,3 (2021): 249-265.
- Whiteaker, Jeffrey R et al. (2021) “Targeted Mass Spectrometry Enables Quantification of Novel Pharmacodynamic Biomarkers of ATM Kinase Inhibition.” Cancers vol. 13,15 3843.
Ordering information
Selection guide for Dynabeads products
Dynabeads products are available for a multitude of molecular and cellular applications. Don’t know where to start? Use our easy selection guide that considers sample type, target molecule, and downstream application to provide tailored recommendations for Dynabeads products.
Immunoprecipitation related content
Literature
Resources
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