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. Use of magnetic beads is one method of isolating your protein of interest via IP. Dynabeads, DynaGreen, and Pierce magnetic beads for IP offer exceptional balance of capacity/yield, reproducibility, purity, and cost for smaller-scale isolation of specific proteins (e.g., IP) and protein complexes (co-IP).
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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.
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 <80min. Pierce protocol finishes in <120min.
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 1). 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.
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.
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.
The protein can be eluted off the beads by using either a mild elution procedure or a denaturing elution procedure (Figure 2).
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:
|Protein A, G, A/G||Secondary antibodies|
|Recommended products||Beads only:||Beads only:|
|Binding properties||Non-covalent antibody binding|
|Antibody co-eluted off the beads||Yes*|
No (Pierce Crosslink Magnetic IP/Co-IP Kit)
|Type of ligand||Different ligands bind different species and antibody subclasses with different specificity†|
|Mass spec compatible||Yes||Yes|
No (Dynabeads M-280)
* 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.
Figure 3. 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.
Figure 4. Electron microscopy of Dynabeads magnetic beads compared to competitor magnetic beads. (A) Dynabeads magnetic beads have a defined surface to carry out the necessary binding with no inner surface to trap any unwanted proteins. 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 non-specific binding.
Figure 5. 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.
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.
Main advantages of magnetic beads for biotin-binding:
Different ligands (antibodies, proteins, etc.) require different bead surface properties and immobilization chemistries. Choose surface-activated beads to control factors such as:
KingFisher instruments automate the isolation of DNA, RNA, proteins, exosomes, viruses, and cells. KingFisher instruments help eliminate cross contamination, produce reproducible results, and cut your hands-on time down to 40 minutes. Set up plates, press start, and walk away.
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:
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.
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.
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