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General

  • In immunoprecipitation (IP), an antibody is used to purify its specific target, or antigen from a mixture.
  • In co-immunoprecipitation (Co-IP), an antibody is used to purify its target antigen, along with its binding partners, from a mixed sample. In this case, the antigen is the bait protein and its binding partners are the prey proteins that are co-purified by the antibody:antigen interaction.
  • Similar to co-IP, a pulldown assay uses a bait protein to “pull down” prey proteins, which are its binding partners. Pulldown differs from IP or Co-IP in that it is not based on an antigen-antibody interaction. Here, the bait protein is immobilized through a tag rather than antibody. Some examples of tags commonly found on a bait protein include: 6x histidine, GST, and biotin. Some examples of binding ligands (on bead) include: nickel/cobalt, glutathione, and streptavidin, respectively, for the stated protein tags.

Magnetic beads are best suited for the routine smaller-scale isolation of specific proteins and protein complexes, providing a balance of capacity/yield, reproducibility, purity and cost savings. Sepharose™ and agarose beads are recommended for purifying large amounts of protein or antibodies.

Direct method

In the direct method, the primary antibody is first bound to the Protein A, G or A/G beads. The beads and sample are then mixed and incubated at 2–8˚C with tilting and rotation. Incubation time will depend upon the concentration of target protein and the specificity of the antibody toward this target. When the concentration of target protein is high, a short incubation time (10 minutes) should be sufficient for target capture. Incubation can be prolonged, up to 1 hour, or even overnight at 4˚C, if necessary.

Indirect method

This is the preferred method when the antibody has poor affinity for the target or when the target protein is in low abundance. The indirect method requires minimal co-incubation of Protein A, G or A/G beads with the sample and allows for optimal control of background binding.

The primary antibody is first incubated with the cell lysate to form an antibody-antigen complex. The complex is then captured by adding Protein A, G or A/G beads to the sample, followed by magnetic separation, or centrifugation in the case of agarose beads. Care must be taken to avoid using an excess amount of antibody, as free antibody will bind to the beads much faster than will the antibody-antigen complex. Free antibody occupying binding sites on the beads may reduce protein yield.

Depending on your specific immunoprecipitation application and location of the target antigen within the cell (i.e., nucleus, cytosol, membrane, etc.,), we offer a variety of different lysis buffers, depending on the sample type and target. You should develop an appropriate lysis strategy to maximize structural integrity. The ionic strength (salt concentration), choice of detergent, and pH of the lysis buffer may affect the immunoprecipitation efficiency as well as the integrity of the target antigen. Some common lysis buffers used include RIPA buffer and NP-40 buffer.

Agarose Immunoprecipitation , Co-Immunoprecipitation, and Pulldown

Crosslinked beaded agarose is the most prevalent type of beaded support used in immunoprecipitation that can be modified for activation or coupled to an appropriate ligand. Agarose is durable and robust, able to withstand centrifugation up to 5,000 x g, pressures of 100 psi, and temperatures up to 120 degrees C without significant loss of structure or flow rate. Agarose exhibits low nonspecific binding in complex samples and is not harmed by moderate levels of most detergents, salts, most organic solvents, or extreme pH. Please see this Tech Tip to read more about immunoprecipitation.

Please see the table below comparing the main highlights, advantages and disadvantages of each kit:

Kit

Highlights

Advantages

Disadvantages

Pierce™ Classic IP Kit

-Antibody bound onto beads via Protein A/G Plus

 

-Used to purify antigen from mixture of proteins

 

-Antibody co-elutes with antigen

-High yield of antigen

 

-Fast protocol

 

-Can be used with purified or crude antibody preparation

-Antibody cannot be reused

 

-Antibody chains may obscure antigen in SDS- PAGE or western blotting

Pierce™ Crosslink IP Kit

-Antibody bound onto beads via Protein A/G Plus

 

-Amine-reactive DSS crosslinker used to covalently link the antibody to the Protein A/G Plus

 

-Used to purify antigen from mixture of proteins

 

-Antibody does not co-elute with antigen

-Moderate yield of antigen

 

-Immobilized antibody can be reused

 

-Can use purified or crude antibody preparations

 

-Antibody chains do not co- elute

-DSS crosslinking may require dose optimization

 

-Antibodies with amines in antigen-binding site can lose functionality due to reaction with DSS crosslinker

Pierce™ Direct IP Kit

-Antibody reacted directly with AminoLink™ bead (no need for Protein A/G) to form covalent bond

 

-No need for DSS crosslinker so no risk of under/over crosslinking

 

-Used to purify antigen from mixture of proteins

 

-Antibody does not co-elute with antigen

-Moderate/high yield of antigen

 

-Immobilized antibody can be reused

 

-Antibody chains do not co- elute

-Needs purified antibody free of extraneous amine groups including carrier proteins

Read more about our kits and select the one best for your experiments on our website.

Magnetic Bead-based Immunoprecipitation and Co-Immunoprecipitation

Magnetic beads, unlike agarose beads, are solid and spherical, and antibody binding is limited to the surface of each bead. While magnetic beads do not have the advantage of a porous center to increase the binding capacity, they are significantly smaller than agarose beads (1 to 4 μm), which collectively gives them adequate surface area-to-volume ratios for optimum antibody binding. 

High-power magnets are used to localize magnetic beads to the side of the incubation tube and out of the way to enable cell lysate aspiration without the risk of also aspirating immune complexes bound to the beads. Magnetic separation avoids centrifugation, which can break weak antibody-antigen binding and cause loss of target protein. 

However, an issue with the use of magnetic beads is that bead size variations may prevent all beads from localizing to the magnet. Additionally, while immunoprecipitation using agarose beads only requires standard laboratory equipment, the use of magnetic beads for immunoprecipitation applications requires high-power magnetic equipment that can be cost-prohibitive. Read more about our Magnetic Immunoprecipitation Products. 

Please see the table below comparing the main highlights, advantages and disadvantages of each kit:

Kit

 

Highlights

Advantages

Disadvantages

Pierce™ Classic Magnetic IP/Co-IP Kit

-Antibody bound onto beads via Protein A/G Plus

 

-Used to purify antigen from mixture of proteins

 

-Antibody co-elutes with antigen

 

-High yield of antigen

 

-Fast protocol

 

-Can be used with purified or crude antibody preparation

-Antibody cannot be reused

 

-Antibody chains may obscure antigen in SDS-PAGE or western blotting

Pierce™ Crosslink Magnetic IP/Co-IP Kit

-Antibody bound onto beads via Protein A/G Plus

 

-Amine-reactive DSS crosslinker used to covalently link the antibody to the Protein A/G Plus

 

-Used to purify antigen from mixture of proteins

 

-Antibody does not co-elute with antigen

 

-Moderate yield of antigen

 

-Immobilized antibody can be reused

 

-Can use purified or crude antibody preparations

 

-Antibody chains do not co-elute

-DSS crosslinking may require dose optimization

 

-Antibodies with amines in antigen-binding site can lose functionality due to reaction with DSS crosslinker

 

Pierce™ Direct Magnetic IP/Co-IP Kit

-Antibody reacted directly with AminoLink™ bead (no need for Protein A/G) to form covalent bond

 

-No need for DSS crosslinker so no risk of under/over crosslinking

 

-Used to purify antigen from mixture of proteins

 

-Antibody does not co-elute with antigen

After Streptavidin-coupled Dynabeads Magnetic Beads for IP and Co-IP section, the Surface-Activated 

Dynabeads Magnetic Beads for IP and Co-IP section with 4 Q/As is missing. Please add these as shown below:

 

-Moderate/high yield of antigen

 

-Immobilized antibody can be reused

 

-Antibody chains do not co-elute

-Needs purified antibody free of extraneous amine groups including carrier proteins

Dynabeads™ Magnetic Beads- Properties

Dynabeads™ magnetic beads are uniform, non-porous, superparamagnetic, monodispersed and highly crosslinked polystyrene microspheres consisting of an even dispersion of magnetic material throughout the bead. The magnetic material within the Dynabeads™ magnetic beads consists of a mixture of maghemite (gamma-Fe2O3) and magnetite (Fe3O4). The iron content (Fe) of the beads is 12% by weight in Dynabeads™ magnetic beads M-280 and 20% by weight in Dynabeads™ magnetic beads M-450. The Dynabeads™ magnetic beads are coated with a thin polystyrene shell which encases the magnetic material, and prevents any leakage from the beads or trapping of ligands in the bead interior. The shell also protects the target from exposure to iron while providing a defined surface area for the adsorption or coupling of various molecules.

Uniformity of bead size and shape provides consistent physical and chemical properties. These uniform physical characteristics lead to high-quality, reproducible results.

The Dynabeads™ magnetic beads are available in three different sizes: 4.5 μm (M-450 beads), 2.8 μm (M-270/M-280 beads), and 1 μm (MyOne™ beads).

The density of these beads is a challenging property to determine. The reason is that Dynabeads™ magnetic beads have a 17–37% magnetic iron oxide content in order to have a reasonable magnetic separation time, and the density of the iron oxide is about 4.9. Dynabeads™ magnetic beads are composite materials, being a mix of polymers and iron oxide, and there are very few polymers that have a density below 1.

The sedimentation rate depends on the bead diameter squared, so the sedimentation of a 1 μm bead is much slower than one of 2.8 μm. The effect of diameter on sedimentation rate is to some extent counteracted by the fact that smaller beads need to have a higher content of iron oxide for magnetic separation applications. Typically, our 280 beads (diameter 2.8 μm) have a density of 1.4 and our 1 μm beads a density of 1.8.

Increasing the density of the water phase should also decrease the sedimentation rate.

Dynabeads™ magnetic beads, being magnetic in nature are really not designed to be centrifuged. That being said, the beads themselves are compact, as the pores in the polymer matrix are filled with magnetic material and coated with a final outer polymer shell that will further add to the rigidity of the beads. Hence, pressure should theoretically not be a problem for the beads themselves, but the force exerted by the beads on surrounding cells in the pellet may be detrimental to the cells.

Two important factors for all solvents are concentration and incubation time. The beads themselves are compatible with a number of solvents (listed below):

  • Ethanol (70% is actually used to sterilize the beads/washings)
  • Methanol
  • Acetone
  • Isopropanol
  • Diglyme
  • DMSO (dimethyl sulfoxide)
  • DMF (dimethylformamide)
  • THF (tetrahydrofuran)
  • Acetonitrile

Yes, they are compatible with 6–8 M Urea.

No. Not only is dithionite a reducing agent, but the strong affinity of the dithionite ion for bivalent and trivalent metal cations (M2+, M3+) allows it to enhance the solubility of iron, making it a chelating agent. As a result, the iron in the Dynabeads™ magnetic beads is reduced and pulled out when they are exposed to dithionite. The same is observed if Dynabeads™ magnetic beads are exposed to DTT and EDTA.

Dynabeads™ Magnetic Beads for Immunoprecipitation and Co-Immunoprecipitation

Using Dynabeads™ magnetic beads for protein isolation provides several advantages:

  • Rapid binding kinetics: since the number of beads per volume for Dynabeads™ is approximately 1,000 times higher than for the same volume of a Sepharose™ slurry, the probability for Dynabeads™ magnetic beads to hit the target is far greater.
  • Incubation time: due to the rapid binding kinetics, the protocol is usually very short.
  • Low background: due to the rapid binding kinetics and the short incubation time, the background is also very low.
  • Trapping of impurities: the beads offer no internal volume for binding or trapping of impurities.
  • Low antibody consumption: this is because Dynabeads™ magnetic beads are nonporous, uniform superparamagnetic, monodispersed, highly crosslinked polystyrene microspheres consisting of an even dispersion of magnetic material throughout the bead. The beads are coated with a thin layer of a highly crosslinked polystyrene shell that encases the magnetic material and prevents any leakage from the beads or trapping of ligands in the bead interior. The outer layer also provides a defined surface area for the adsorption or coupling of various molecules such as antibodies. Uniformity of bead size and shape provide consistent physical and chemical properties. These uniform physical characteristics lead to high-quality, reproducible results.
  • Reproducibility: due to easier practical handling, such as pipetting. No centrifugation steps or preclearing are required.

The antibody binding capacity of a Sepharose™ slurry is higher, but for researchers doing immunoprecipitation, this should not be a concern. Binding capacity in immunoprecipitation is much more dependent on the amount of antibody used rather than the binding capacity of the beads or slurry, and the excess binding capacity of a Sepharose™ slurry in immunoprecipitation will just contribute to increased nonspecific binding or waste of precious antibody.

However, if the application is targeted at large-scale antibody purification rather than analytical immunoprecipitation, the capacity of the Sepharose™ slurry may become an advantage over Dynabeads™ magnetic beads. Hence, we are cautious in recommending Dynabeads™ magnetic beads for preparative applications.

There are two main ways of coupling antibodies to the beads (with several different options within the two groups): covalent coupling and noncovalent coupling.

For covalent coupling, there are several choices including:

  • Dynabeads™ M-280 Tosylactivated magnetic beads (Cat. No. 14203)
  • Dynabeads™ M-270 Epoxy magnetic beads (Cat. No. 14301)
  • Dynabeads™ Antibody Coupling Kit (Cat. No. 14311D, Dynabeads™ M-270 Epoxy and buffers for covalent coupling)

For co-immunoprecipitation, we offer the Dynabeads™ Co-Immunoprecipitation Kit, Cat. No. 143-21D, containing Dynabeads™ M-270 Epoxy magnetic beads, buffers for covalent coupling, and buffers optimized for co- immunoprecipitation (see Alber F, Dokudovskaya S, Veenhoff LM et al. (2007) Determining the architectures of macromolecular assemblies. Nature  450(1710):683–694).

Dynabeads™ magnetic beads can also be used for immunoprecipitation, which relies on noncovalent binding of antibodies. The most common products used for noncovalent binding of antibodies to Dynabeads™ magnetic beads are:

  • Dynabeads™ Protein A magnetic beads (Cat. No. 10001D)
  • Dynabeads™ Protein G magnetic beads (Cat. No. 10003D)
  • Immunoprecipitation Kit—Dynabeads™ Protein A magnetic beads (Cat. No. 10006, containing Dynabeads™ magnetic beads and optimized buffers for immunoprecipitation)
  • Immunoprecipitation Kit—Dynabeads™ Protein G magnetic beads (Cat. No. 10007, Dynabeads™ magnetic beads and optimized buffers for immunoprecipitation)
  • Dynabeads™ magnetic beads coated with secondary antibodies. (Dynabeads™ Pan Mouse IgG magnetic beads (Cat. No. 11041) are 4.5 μm beads developed for cell separation that have increased capacity per volume when using Dynabeads™ M-280 Sheep Anti-Mouse IgG magnetic beads.

The Dynabeads™ Co-Immunoprecipitation Kit (Cat. No. 14321D) was developed to mimic the intracellular conditions while providing customized stringency for different protein complexes in a cell solution. It offers the opportunity to optimize and fine-tune weak and transient interactions between short amino acid sequences in different proteins.

The beads and buffers provided in the Dynabeads™ Co-Immunoprecipitation Kit will enable you to:

  • Covalently immobilize antibodies of your choice onto the surface of Dynabeads™ M270 epoxy magnetic beads
  • Use the antibody coated beads for co-immunoprecipitation 

Benefits of the kit:

  • Freedom to use any antibody
  • No release of antibody in the eluate
  • Co-IP buffer system
  • Easy optimization
  • Qualitative reproducibility

The most optimal antibody concentration for coupling is below 10 μg/mg Dynabeads™ magnetic beads and provides efficient coupling and functionality of the coupled antibody. Presence of 0.1% NaN3 and/or 0.1% BSA does not reduce coupling efficiency and functionality of the antibodies.

The Dynabeads™ Co-Immunoprecipitation Kit contains Dynabeads™ M-270 Epoxy beads and proprietary buffers for co- immunoprecipitation.

The lysis buffer in the Dynabeads™ Co-immunoprecipitation Kit will not lyse the nuclei; however, the IP Lysis Buffer in the Pierce™ IP and Co-IP Kits (Agarose and Magnetic) will lyse both the cell membrane and the nuclear membrane.

The Dynabeads™ Antibody Coupling kit includes buffers, beads, and the protocol for covalent antibody coupling to beads. The Dynabeads™ Co-Immunoprecipitation kit includes the complete Antibody Coupling Kit, plus the additional buffers and protocol required for successful co-immunoprecipitation.

The amount of beads required would depend on the downstream analysis you will be performing. For instance, using 1.5 mg coated beads with 0.05–1 g of total cell extract is recommended if the downstream experiment is western blotting. For mass spectrometry, more beads are required (typically 7.5 mg beads for 1–15 g of total cell extract). In cases where the target is purified, you will probably need fewer beads. The amount might also depend on whether total depletion of the main protein is required.

If the volume of antibody is equal to (or exceeds) the volume of C1, the antibody solution can be used directly (as long as the pH is approx. 7.4). So, for 5 mg Dynabeads™ magnetic beads, add 250 μL antibody + 250 μL C2.

Coupling of large amounts of antibody (20 to 30 μg antibody/mg beads) is difficult. If loading 20 μg of antibody to the beads, the total amount of antibody covalently coupled to the beads will not be more than 13 μg/mg beads. Up to 10 μg, you will find a linear relationship between added antibody and coupled antibody. Above these concentrations, the antibody will precipitate down to the bead surface but a reduction in covalent bonds will be observed.

The recommended amount of antibody for a coupling reaction is typically 5–7 µg antibody/mg Dynabeads™ magnetic beads.

If the target protein has the same molecular weight as the heavy or light chain antibody, we would recommend covalently binding the antibody to the bead surface. This can be done by either crosslinking the antibody to the Dynabeads™ Protein A or G magnetic beads, or secondary coated beads, or by using one of the surface-activated Dynabeads™ magnetic beads.

Dynabeads™ magnetic beads coated with antibody/ligand may be stored at 2–8 degrees C without loss of antigen binding capacity. For long-term storage, a final concentration of 0.02% NaN3 may be added to the antibody coupled beads in a physiological buffer. Please note, not all coupled antibodies retain their function in long term storage. Verify your coupled antibody stability by testing in small scale. After storage, coated Dynabeads™ magnetic beads should be washed once in PBS/BSA for 5 min before use.

Incubation time will depend on the immunogenicity of the primary antibody and its binding affinity with the specific antigens. For a good primary antibody, 30–40 minutes incubation should work well. If you are working with a poor antibody or a very low-abundance protein, you could try to increase binding by incubating overnight. However, this also increases the chance of background protein binding

There are several methods to quantify the amount of antibody bound to the beads. The crudest method is to measure (using fluorescence or even OD) the concentration of antibody in the coupling supernatant before and after conjugation. Alternatively, you could measure the amount of antibody bound to the beads by fluorescence, chemiluminescence, or radiolabeling detection methods.

Yes, but this would require some optimization of the coupling process since the two ligands may have inherent differences in hydrophobic properties and isoelectric points that would, in turn, influence the coupling conditions (pH, temperature, etc.). If a particular ratio of the two ligands on the Dynabeads™ magnetic beads surface is desired, you will need to test the binding capacity for each ligand separately and then optimize the coupling process for the combination of ligands. For coupling more than one ligand, we recommend the Dynabeads™ M-270 Epoxy magnetic beads—either the stand-alone beads, Cat. No. 14301 (coupling buffers are not included) or Dynabeads™ Antibody Coupling kit, Cat. No. 14311D (coupling buffers are included).

Within practical limits, elution volume can be scaled up or down to suit your experiment. Volumes less than 10 μL become more difficult to work with. In addition, the amount of target is important. If you have a lot of beads bound by a lot of target in a small elution volume, your elution may not be very efficient. Typically, 15–100 μL of beads may be eluted in 30 μL. For efficient recovery of the antigen and/or binding partners, the elution volume should at minimum equal the volume of the beads.

Dynabeads™ M-450 magnetic beads are 4.5 micron beads and may be used for immunoprecipitation. An example is Dynabeads™ Sheep anti-Rat IgG magnetic beads (Cat. No. 11035), which you may use when your primary antibodies are of rat origin. The smaller beads (2.8 micron) do, however, provide larger surface area and will give higher yields of protein.

The Dynabeads™ Co-immunoprecipitation Kit is specially designed for protein complex pulldown only, not for simple IP. These beads are the Dynabeads™ M270 Epoxy (Cat. No. 14301) beads and are used for covalent binding of the antibody so it will not be co-eluted off with the target complex during mild elution. The kit also contains a buffer system that should make it easier to optimize for the complex.

With the Dynabeads™ Protein A or G magnetic beads, there is only an affinity binding between the beads and the antibody and hence the antibody will be co-eluted with the target protein unless you crosslink it to the beads. Hence, in general, we recommend the Dynabeads™ Co-immunoprecipitation Kit for complex pulldown, especially if you are working with large, labile complexes or are doing downstream mass spectrometry.

Secondary coated Dynabeads™ magnetic beads (like the Dynabeads™ anti-mouse IgG beads) can also be used for IP, but, the drawback when compared to Dynabeads™ Protein A or G magnetic beads, is that it will only work with one species of antibody. However, the binding between the primary antibody and the secondary antibody might be a bit stronger than between Protein A or G and the antibody. Hence, you can apply more stringent washing conditions. That being said, Dynabeads™ Protein A and G magnetic beads should give a very low background.

Answering this question is not straightforward. It will depend on the detection method. When using HRP (horseradish peroxidase–based detection system or radioactivity in combination with a good antibody, very little target is required. More target is required when using an AP (alkaline phosphatase)–based detection system. When a sensitive detection system is used, detection will most likely be in the range of nanograms and possibly pictograms of target.

Dynabeads™ Protein A and Dynabeads™ Protein G Magnetic Beads for IP and Co-IP

Here are the advantages of using Dynabeads™ Protein A or Dynabeads™ Protein G magnetic beads:

  • Perfect for small-scale IgG purification and immunoprecipitation
  • Require no columns, centrifugation, or time-consuming pre-treatment of samples
  • Ensure easy handling and a simple protocol (workflow)
  • Are gentle, placing minimal physical stress on precious proteins
  • The starting sample can be saliva, plasma, ascites, serum, and tissue culture or hybridoma supernatants
  • The recombinant Protein A or G on the beads contains no albumin binding sites, thus avoiding co-purification of contaminating proteins

The Dynabead™ Protein A and G magnetic beads are 2.8 micron in size.

We have tested stability of samples that have been stored for two weeks at 25 or 37 degrees C and then moved to 2 to 8 degrees C. The results of these tests show that storage of Dynabeads™ magnetic beads at up to 37 degrees C for two weeks does not adversely affect either the immediate test result or the stability of the product. Coated Dynabeads™ magnetic beads can usually be stored for several months at this temperature, depending on the stability of the immobilized ligand.

Glycerol most likely inhibits nonspecific hydrophobic interactions and therefore high concentrations of glycerol might affect the affinity binding of IgGs to Protein A or G. If the antibodies are stored in glycerol, the glycerol concentration is most likely high. We would suggest dialyzing the antibody solution. If the antibody concentration is high, the antibody- glycerol could be diluted instead by following the protocol, and this dilution may be enough to avoid dialysis. This would need to be tested empirically.

Yes. Elution of isolated proteins without release of the specific antibodies, and reuse of the immobilized antibodies requires covalent crosslinking. Protein A and Protein G are supplied covalently bound to the beads. Crosslinking the antibodies bound to Dynabeads™ Protein A or Dynabeads™ Protein G magnetic beads will result in covalent binding of the antibodies to Protein A/G. Commercially available crosslinkers reacting with protein amine groups can be used. Optimization may be required.

The procedure described below is for the isolation of Igs from a 100 μL sample containing between 0.2 μg (2 μg/mL) and 250 μg (2.5 mg/mL) Igs. The protocol can be scaled up and down as required.

Washing Procedure

  1. Resuspend the Dynabeads™ Protein G thoroughly to obtain a homogeneous suspension.
  2. Transfer the desired volume of Dynabeads™ Protein G to a tube at room temperature. In order to isolate Ig from a 100 μL sample, it is generally recommended to use 20–100 μL of the Dynabead™ Protein G. A higher volume can be used if the sample has a high Ig concentration.
  3. Place the tube on the magnet for 1 min and discard the supernatant by aspiration with a pipette while the tube remains on the magnet.
  4. Remove the tube from the magnet, add 0.5 mL of a citrate-phosphate buffer*, pH 5.0 and resuspend the Dynabeads™ Protein G.
  5. Repeat steps 3, 4 and 3.

Ig Capture Procedure

  1. Add 100 μL sample containing Igs to the washed Dynabeads™ Protein G.
  2. Incubate with gentle mixing for 40 min at room temperature.
  3. Place the test tube on the magnet for 2 min and discard the supernatant.
  4. Remove the test tube from the magnet and add 0.5 mL citrate-phosphate buffer, pH 5.0. (For downstream immunoprecipitation or storage of Dynabeads™ Protein G, 0.01_0.1% Tween™-20 can be added to the buffer to prevent aggregation).
  5. Wash the Dynabeads™ Protein G by repeating steps 3 and 4 twice.
  6. Place the test tube on the magnet for 2 mins and discard the supernatant. The captured Igs are now ready to be eluted off the Dynabeads™ Protein G, or the Dynabead™ Protein G-Ig complex can be further used for immunoprecipitation.

Ig Elution Procedure

Elution of Igs is, in this example, performed by lowering pH using 0.1 M citrate (pH 2–3). The degree of acidity needed depends on the species and Ig subclass, but at pH 3.1 most Igs will be eluted off.

  1. Add 30 μL 0.1 M citrate (pH 2-3) to the Dynabeads™ Protein G-Ig complex.
  2. Mix well by tilting and rotation for 2 mins.
  3. Place the test tube on the magnet for 1 min and transfer the supernatant containing purified Igs to a new tube.
  4. Repeat step 1, 2, and 3 in order to elute any remaining Ig. Pool the supernatants containing the pure Igs (total collected volume = 60 μL).

*Washing Buffer Recipe:

To make the citrate-phosphate buffer, pH 5, mix 4.7 g citric acid (MW=192) with 9.2 g dibasic sodium phosphate (Na2HPO4) dehydrate (MW=178). Fill up to 1 L with distilled water.

Please note, in the protocol we recommend using citrate-phosphate buffer pH 5.0; however, it is also possible to use other buffers like 0.1 M Na-citrate pH 5.0 or 0.1 M Na-acetate pH 5.0.

Here is a method for crosslinking of IgG immobilized Dynabeads™ Protein G magnetic beads (5 μg goat a-HSA immobilized to 50 μL Dynabeads™ Protein G magnetic beads, in PBST), with BS3 linker (Cat. No. 21580):

  1. BS3 Stock and Conjugation solutions have to be freshly prepared just before use (i.e., make them after IgG immobilization is complete).
  2. Prepare 100 mM BS3 in Conjugation Buffer (Stock Solution). Proceed to making a 5 mM solution by diluting in Conjugation Buffer.
  3. Wash the Ig-coupled Dynabeads™ Protein G magnetic beads twice in 200 μL Conjugation Buffer. Place on magnet and discard supernatant.
  4. Resuspend the Dynabeads™ magnetic beads in 250 μL 5 mM BS3.
  5. Incubate for 30 min with tilting/rotation at room temperature.
  6. Quench the crosslinking reaction by adding 12.5 μL Quenching Buffer
  7. Incubate for 15 min with tilting/rotation at room temperature.
  8. Wash crosslinked Dynabeads™ magnetic beads three times with 200 μL PBST (or IP buffer of your choice), place on magnet, discard supernatant
  9. Proceed with your IP and antigen elution

BS3 Conjugation buffer: 20 mM sodium phosphate, 0.15 M NaCl, pH 7–9.
BS3 Quenching buffer: 1 M Tris HCl, pH 7.5.

The Dynabeads™ Protein A and G magnetic beads contain hydrophilic beads that are coupled with Protein A or Protein G respectively. The hydrophilic surface is not blocked with BSA.

Blocking with BSA will work best on a hydrophobic surface, where surface adsorption keeps the blocker in position. Dynabeads™ Protein A and G magnetic beads have a hydrophilic surface. Hence, BSA blocking may not be very successful, as surface adsorption is not promoted between the hydrophilic surface and the hydrophobic BSA. Instead, reduce nonspecific binding by performing more stringent washing and adding Tween™ 20 detergent (concentrations of 0.01–0.1%) to the washing buffer.

Use mild elution conditions, e.g., a buffer with high salt or low pH. Heating the beads at 95 degrees C for 5 minutes in SDS buffer will elute the antibody as well.

Using Dynabeads™ Protein G (or Dynabeads™ Protein A magnetic beads) alone with your IP sample is not a good control. Different molecules in your sample will bind either to Protein G (or Protein A) or to the beads themselves through a variety of interactions (hydrophobic interactions, charge interactions etc.). As a negative control, you may use Dynabeads™ Protein G (or Dynabeads™ Protein A magnetic beads) bound to a non-related IgG.

The Wash and Bind buffer is 0.1 M NaPhosphate and 0.01% Tween™-20 at a pH of 8.2.

Please see the table below:

Ig origin

Affinity for Protein A

Affinity for Protein G

Human IgG1,2,4

+++

+++

Human IgD

Human IgA, E, M

+

Human IgG3

+

+++

Mouse IgG1

+

+++

Mouse IgG2a, 2b, 3

+++

+++

Mouse IgM

+

+

Rat IgG1

+

+

Rat IgG2a

+++

Rat IgG2b

+

Rat IgG2c

+++

+

Bovine IgG1

+

+++

Bovine IgG2

+++

+++

Chicken IgY

Dog IgG

+++

+

Goat IgG1

+

+++

Goat IgG2

+++

+++

Guinea pig IgG

+++

+

Hamster

+

NA

Horse IgG

+

+++

Monkey IgG

+++

+++

Porcine IgG

+++

+++

Rabbit IgG

+++

+++

Sheep IgG1

+

+++

Sheep IgG2

+++

+++

Yes, for sensitive proteins or protein complexes, the protocol can be performed at 4 degrees C. Lowering the temperature will slow the binding kinetics, so the incubation time should also be increased.

These beads will bind with different affinities depending on the immunoglobulin type and species. Binding occurs mainly through the Fc region. The binding capacity will depend on the initial concentration in the sample and the source of the antibody. Please see the comparison table below:

 

Dynabeads™ Protein A beads

Dynabeads™ Protein G beads

Source

Bacillus

E. coli

Molecular weight

45 kDa

17 kDa

Number of binding sites for IgG

4

2

Binding capacity

~250 µg human IgG/mL beads

~640 µg mouse IgG/mL beads

Optimal binding pH

7.4

7.4

Due to the very low nonspecific binding properties of Dynabeads™ Protein A and Dynabeads™ Protein G magnetic beads, generally pre-clearing is not required with these kits.

Dynabeads™ Protein A and Dynabeads™ Protein G magnetic beads are excellent for Co-IP/IP. The advantages are:

  • Elimination of background
  • Gentle pulldown
  • Reduced protocol time

Streptavidin-coupled Dynabeads™ Magnetic Beads for IP and Co-IP

M stands for magnetic; M-280 refers to hydrophobic 2.8 micron beads while M-270 refers to hydrophilic 2.8 micron beads. MyOne™ refers to 1 micron beads.

  • One milligram of Dynabeads™ M-280 Streptavidin magnetic beads typically binds 650–900 pmol of free biotin, 200 pmol of biotinylated peptide, up to 10 μg of biotinylated antibody, 10 μg of biotinylated double-stranded DNA, or 200 pmol of biotinylated single-stranded oligonucleotides.
  • One milligram of Dynabeads™ M-270 Streptavidin magnetic beads typically binds more than 950 pmol of free biotin, 200 pmol of biotinylated peptide, up to 10 μg of biotinylated antibody, 10 μg of biotinylated double- stranded DNA, or 200 pmol of biotinylated single-stranded oligonucleotides.
  • One milligram of Dynabeads™ MyOne™ Streptavidin C1 magnetic beads typically binds more than 2,500 pmol free biotin, 400 pmol of biotinylated peptides, up to 20 μg of biotinylated antibody, 20 μg of biotinylated double-stranded DNA, or 500 pmol of biotinylated single-stranded oligonucleotides.
  • One milligram of Dynabeads™ MyOne™ Streptavidin T1 magnetic beads typically binds 1,100–1,700 pmol free biotin, 400 pmol of biotinylated peptides, up to 20 μg of biotinylated antibody, 20 μg of biotinylated double- stranded DNA, or 400 pmol of biotinylated single-stranded oligonucleotides.

The exact number of streptavidin molecules bound per bead is not measured, but is approximately 14–16 µg streptavidin per mg Dynabeads™ M-280 Streptavidin magnetic beads.

Streptavidin is a protein made up of four identical subunits, each containing a high affinity binding site for biotin (KD = 10–15 M). Streptavidin has the same biotin binding properties as avidin, but less nonspecific binding is observed. After immobilization on the beads, there are 2–3 binding sites free for interaction with biotin.

The streptavidin molecule is covalently attached to the surface of the beads; under normal, recommended conditions, negligible leakage is detected (less than 0.2% of total attached streptavidin after 2 months at 37 degrees C). However, it should be noted that not all of the four streptavidin subunits are covalently coupled to the beads. Typically, one or two of the subunits are covalently coupled. Streptavidin is like other proteins; if heated, it can denature and dissociate into subunits. If Dynabeads™ Streptavidin magnetic beads are, for instance, boiled, some of the streptavidin subunits may be released (as monomers or aggregates) from the beads. The covalently bound streptavidin subunits will not be affected by such treatment. When streptavidin is bound to biotin, the streptavidin-biotin complex is more stable than the unbound streptavidin molecule.

Which product to choose depends on the properties of your sample, the buffers and solutions applied, as well as downstream application. In general all the Dynabeads™ Streptavidin beads can be used in applications involving biotinylated ligands, however, some beads may perform better than the others in particular applications due to their characteristics. Please refer to the Dynabeads™ Streptavidin Selection Guide for detailed information.

We do not recommend this as streptavidin becomes hydrophobic and aggregates during denaturing.

The streptavidin-biotin interaction is the strongest known non-covalent, biological interaction between a protein and molecule. The bond formation between biotin and avidin is very rapid and, once formed, is unaffected by wide extremes of pH, temperature, organic solvents, and other denaturing agents. Unless derivative forms of biotin or modified streptavidin have been adopted for your experiment, requiring a specific form and normally gentle way to dissociate biotin from streptavidin, often very harsh methods are required to dissociate the biotin from streptavidin which will irreversibly denature the streptavidin.

For biotinylated proteins, boil the beads in 0.1% SDS or SDS-PAGE buffer for 3 minutes or 8 M guanidinium hydrochloride, pH 1.5.

Use mild elution conditions, e.g., a buffer with high salt or low pH. Heating the beads at 95 degrees C for 5 min in SDS buffer will elute the antibody as well.

Storage should be at 2 to 8 degrees C. Freezing or drying of the Dynabeads™ magnetic beads is not recommended. Provided the Dynabeads™ magnetic beads are stored correctly, quality is guaranteed until the expiry date stated on the label.

Surface-Activated Dynabeads™ Magnetic Beads for IP and Co-IP

  • Dynabeads™ magnetic beads M-280 Tosylactivated allows direct covalent binding to primary amino or sulfhydryl groups in proteins and peptides at high pH and temperature. 
  • Dynabeads™ magnetic beads M-270 Epoxy allows direct covalent binding to primary amino and sulfhydryl groups in proteins and peptides at neutral pH and a wide temperature range. 
  • Dynabeads™ magnetic beads M-270 Amine allows direct covalent binding through reductive amination of aldehydes, or use of bi-functional NH2-reactive crosslinkers. 
  • Dynabeads™ magnetic beads M-270 Carboxylic Acid and MyOne™ Carboxylic Acid allows covalent amide formation with primary amino groups in proteins and peptides. 

This depends on the nature of the specific ligand to be immobilized and the desired downstream application. 

  • The most frequently used surface-activated Dynabeads™ magnetic beads for protein isolation are the Dynabeads™ magnetic beads M-280 Tosylactivated. Dynabeads™ magnetic beads M-280 Tosylactivated are hydrophobic, easy to handle and ideal for covalent binding of antibodies for immunoprecipitation. Other ligands could also be covalently bound to these beads, but they have to tolerate conditions like neutral to high pH and high temperatures (required for covalent bond formation). 
  • Dynabeads™ magnetic beads M-270 Epoxy is used when the ligand to be immobilized needs to be treated gently and will not tolerate harsh binding conditions like high temperature or pH. Proteins/peptides (other than antibodies) and enzymes are often coupled onto these beads. 
  • Dynabeads™ magnetic beads M-270 Amine is often used in combination with crosslinkers to create specific surface groups on the beads. Hetro-bifunctional crosslinkers with an amine binding NHS group at one end and another chemical group of your choice at the other end are most frequently used. For example, Dynabeads™ magnetic beads M-270 Amine can be coated with a hetero-bifunctional crosslinker containing a NHS group at one end and maleimide at the other end to create Dynabeads™ magnetic beads with a maleimide surface. Since maleimide reacts specifically with sulfhydryl groups, these modified Dynabeads™ magnetic beads can be used for applications where binding of sulfhydryl groups are desired. Dynabeads™ magnetic beads M-270 Amine may also be used for direct ligand coupling via aldehyde or ketone groups by Schiff base (imine) formation and reductive amination. In addition carboxylic acid groups on a ligand can be activated with cabodiimide, which results in a direct amide bond formation between the beads and the ligand. Alternatively, a crosslinker may be introduced to the ligand. After activation of the ligand with crosslinker, the free end on the crosslinker has to be amine reactive. 
  • Dynabeads™ magnetic beads M-270 Carboxylic Acid and Dynabeads™ magnetic beads MyOne™ Carboxylic Acid can also be used for immobilizing proteins. The carboxylic acid groups on these beads need to be activated (with carbodiimide) before coupling. The negative surface of these beads may attract positively charged proteins and cause nonspecific binding. This needs to be considered if these beads are going to be used for immunoprecipitation. 

A linker antibody provides proper orientation of the primary antibody, specific for the target. Optimal orientation of the primary antibody is more important for larger organelles than for small organelles or membrane fractions. Different linkers can be used, but we recommend an Fc-binding antibody, such as a monoclonal or polyclonal anti-mouse IgG. The linker antibody must be affinity purified and not contain stabilizers such as sugars or proteins that may bind to the Dynabeads™ magnetic beads. The specific primary antibody, if polyclonal, must be affinity purified in order to provide a high density of the specific antibody on the Dynabeads™ magnetic beads surface. 

We offer tosyl-, epoxy-, carboxylic acid–, and amine-activated Dynabeads™ magnetic beads. See our bead comparison.

Secondary Antibody-Coated Dynabeads™ Magnetic Beads for IP and Co-IP

Our secondary antibody-coated Dynabeads™ magnetic beads are pre-coated with affinity purified polyclonal secondary antibodies. We offer the following secondary antibody-coated Dynabeads™ magnetic beads:

  • Dynabeads™ M-280 Sheep anti-Rabbit IgG (Cat. No. 11204D): 2.8 µm beads to be used when your primary antibody is derived from rabbit serum
  • Dynabeads™ M280 Sheep Anti-Mouse IgG (Cat. No. 11201D): 2.8 µm beads to be used when your primary antibody is derived from mouse
  • Dynabeads™ Sheep anti-Rat IgG (Cat. No. 11035): larger 4.5 µm beads to be used when your primary antibody is derived from rat; please note that smaller beads provide a larger surface area and hence would give higher yields of protein
  • Dynabeads™ Pan Mouse IgG (Cat. No. 11041): 2.8 µm beads that will bind all subclasses of mouse IgG

We offer the following secondary antibody–coated Dynabeads™ magnetic beads:

  • Dynabeads™ M-280 Sheep anti-Rabbit IgG (Cat. No. 11204D): 2.8 µm beads to be used when your primary antibody is derived from rabbit serum
  • Dynabeads™ M280 Sheep Anti-Mouse IgG (Cat. No. 11201D): 2.8 µm beads to be used when your primary antibody is derived from mouse
  • Dynabeads™ Sheep anti-Rat IgG (Cat. No. 11035): 4.5 µm beads to be used when your primary antibody is derived from rat.
  • Dynabeads™ Pan Mouse IgG (Cat. No. 11041): 4.5 µm beads that will bind all subclasses of mouse IgG

Although Dynabeads™ Sheep anti-Rat IgG and Dynabeads™ Pan Mouse IgG may be used for immunoprecipitation, please note that smaller (2.8 µm) beads provide a larger surface area and hence would give higher yields of protein than 4.5 µm beads.

Yes. Elution of isolated proteins without release of the specific antibodies, and reuse of the immobilized antibodies requires covalent crosslinking. Secondary antibodies are supplied covalently bound to the beads. Crosslinking the bound primary antibodies to the secondary antibody-coated Dynabeads™ magnetic beads will covalently bind the primary antibodies to the secondary antibodies. A protocol for crosslinking is available in Dynabeads™ magnetic beads product manuals. However, we cannot guarantee the full recovery of your antibody activity, as this varies from antibody to antibody. Commercially available crosslinkers reacting with protein amine groups can be used. Optimization may be required.

Dynabeads™ His-Tag Isolation and Pulldown

This principle is based on the reversible interaction between various amino acid side chains and immobilized metal ions. Depending on the immobilized metal ion, different side chains can be involved in the adsorption process. Most notably, histidine, cysteine, and tryptophan side chains have been implicated in protein binding to immobilized transition metal ions and zinc. Histidines exhibit highly selective binding to certain metals and have great utility in immobilized metal affinity chromatography (IMAC). Under conditions of physiological pH, histidine binds by sharing electron density of the imidazole nitrogen with the electron-deficient orbitals of transition metals. Although only three histidines may bind transition metals under certain conditions, six histidines reliably bind transition metals in the presence of strong denaturants such as guanidinium. Such protein tags are commonly referred to as 6xHis tags. For isolation of His-tagged proteins, we offer the Dynabeads™ His-Tag Isolation & Pulldown beads (Cat. Nos. 10103D, 2 mL (40 mg beads/mL) and 10104D, 10 mL (40 mg beads/mL)). These beads have a cobalt-based Immobilized Metal Affinity Chromatography (IMAC) surface chemistry. The technology is comprised of a tetradentate metal chelator in which four of cobalt's six coordination sites are occupied. The imidazole rings of histidine residues present in a poly histidine peptide chain are able to occupy the two remaining coordination sites, resulting in protein binding.

For isolation of His-tagged proteins, we offer the Dynabeads™ His-Tag Isolation & Pulldown beads (Cat. Nos. 10103D, 2 mL (40 mg beads/mL) and 10104D, 10 mL (40 mg beads/mL)).

Binding Capacity: approximately 40 μg His-tagged GFP per milligram of beads

Purity: close to 100% (assessed by Bioanalyzer™ instrument)

DTT, DTE, EDTA, and EGTA are not compatible with Dynabeads™ His-Tag Isolation & Pulldown magnetic beads. You should make sure that these are not present. In addition, we have some evidence that beta-mercaptoethanol and MgCl2 may have an impact on the cobalt as well.

BSA would only prevent nonspecific adsorption to the surface. Also, if the BSA binds to the chelator, it will block His-tag binding. Instead, we recommend that you use 0.01% Tween™ 20 detergent instead.

Using an EDTA solution (dissolved in water) should work, but the elution efficiency may be low due to interactions between the beads and target. You may use detergent or salt to reduce this effect, or use low pH (or low pH in combination with denaturing conditions) to increase elution efficiency.

Alternatively, EDTA may be dissolved in 10–100 mM Binding and Wash buffer (B&W buffer). Since the B&W buffer contains Tween™ detergent, proteins eluted using this solution should be gel purified if downstream mass spectrometry is to be performed.

DynaMag™ Magnets

The DynaMag™-2 Magnet (Cat. No. 12321D) can hold 16 standard 1.5 mL or 2 mL microcentrifuge tubes, while the DynaMag™-Spin Magnet (Cat. No. 12320D) holds up to 6 standard 1.5 mL microcentrifuge tubes.

Spray and/or wipe the DynaMag™ unit with one of the following cleaning agents:

  • 70% isopropyl alcohol
  • 1% sodium hypochlorite solution (bleach)
  • 0.1 N HCL solution

Do not submerge the unit in aqueous solutions and avoid prolonged exposure to water or solutions. Do not autoclave or heat the magnet above 70 degrees C.

1 Tesla is equivalent to 10,000 Gauss.

The DynaMag™-2 Magnet is 3,500–3,700 minimum Gauss level and the DynaMag™-Spin Magnet is 3,000–3,500 minimum Gauss level.

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