Dynabeads™ Protein G - for oem / Industrial use only - FAQs

Here are a few suggestions to try if you are getting low binding to the Dynabeads protein A/G beads:

-Verify the binding/specificity of your antibody to your antigen, e.g., by ELISA
-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
-If you have used the indirect IP method, try the direct IP method; conversely, if you have used the direct IP method, try the indirect IP method
-Check the amount of beads and sample volume with respect to the capacity of the different beads provided in the package inserts, and increase the amount of beads or the concentration of your antibody during coupling
-Increase the incubation time
-If using a commercially sourced antibody, confirm it is validated for IP
-Try another antibody


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Here are a few suggestions you can try:

-Perform the IP without crosslinking the antibody to the beads
-Reduce the amount of crosslinker used to covalently attach the antibody to the beads
-Try a different crosslinker
-To prevent co-elution of antibody, try one of our surface-activated Dynabeads magnetic beads; this allows you to conjugate the antibody to the beads directly, through covalent binding

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Here are some suggestions:

-Wash the beads prior to use in 100 mM glycine at a pH of 11.3 followed by a wash with 200 mM glycine at a pH of 2.8 for a very short period. Immediately transfer the beads to PBS with 0.01% Tween-20.
-Perform the elution in the low pH, glycine-based elution buffer recommended in the manual.
-Crosslinking of the antibody to the Protein G also crosslinks the Protein G to itself, which anchors it to the bead surface.

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Protein G is coated onto hydrophilic beads. If your background is protein-mediated, then we normally suggest having a combination of blocking protein and non-ionic detergent both in the coupling and washing buffer to reduce nonspecific binding

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The binding sites of your antibody have likely been altered by the crosslinking. When this occurs, your antibody will show reduced affinity or no affinity to its target antigen. Another consequence of crosslinking can also be increased affinity for unintended (nonspecific) targets. This is always a high risk with crosslinking, and it is a problem easily avoided by choosing another path to covalent antibody coupling. You can try using the Dynabeads Antibody Coupling Kit. This kit is a far superior solution for covalent antibody coupling to Dynabeads magnetic beads (compared to crosslinking with Dynabeads Protein A or G magnetic beads). The Dynabeads Antibody Coupling Kit is compatible with almost any antibody. It is designed specifically for covalent antibody coupling to Dynabeads magnetic beads. Unlike crosslinking, the Dynabeads Antibody Coupling Kit will not alter antibody specificity or affinity.

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Streptococcal Protein G comprises two or three domains that bind to the constant Fc region of most mammalian immunoglobulin Gs (IgGs). Protein G binds to the interface between the second and third heavy chain constant domains of Fc, which is roughly the same binding site used by Protein A. Protein G comprises one ?-helix packed onto a four- stranded ?-sheet. Residues from Protein G that are involved in binding are situated within the C-terminal part of the ?- helix, the N-terminal part of the third ?-strand and the loop region connecting these two structural elements. The Protein G:Fc complex involves mainly charged and polar contacts, whereas Protein A and Fc are held together through nonspecific hydrophobic interactions and a few polar interactions.

Elution, both mild and denaturing, will destroy the binding between antibodies to Protein A or Protein G. The strong elution also breaks the bond between the heavy and light chains of the antibodies and therefore the antibodies will be seen on the gels. The IgG heavy chain is about 50 kDa, the IgG light chain is about 25 kDa, and the IgG whole complex is about 150 kDa. NOTE: Mild elution does not always break the affinity of IgG to Protein A or Protein G. And strong elutions only break the IgG chains apart if a reducing agent is present.

If you do not want to see the antibody on the gels, you may consider crosslinking the antibody and Protein G or Protein A, prior to the immunoprecipitation. Alternatively, you can also use different species of antibody for blotting to avoid seeing the primary antibodies use for IP. For example, if you use the monoclonal antibody for IP, you can blot the membrane using a polyclonal antibody that would not recognize the monoclonal antibody.

Finally, the Clean-Blot IP Reagent detects only the native IgG; it will not detect IgG that has been denatured in SDS- PAGE Sample Buffer.

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Here are a few suggestions to try:

-Use more stringent conditions for washing
-Add a non-ionic detergent (Tween-20 or Triton X-100) to the washing buffer, at concentrations between 0.01-0.1% (v/v)
-If the beads are blocked before precipitation, add the same blocker to the washing buffer
-Increase the number of washing steps
-Prolong the duration of the washing steps and/or add a soak step
-Decrease the incubation time between the beads and the sample
-Try the indirect IP method (add the antibody to the sample, incubate, then capture the immune complexes with the beads)
-Decrease the antibody concentration
-A pre-clearing step may be performed to remove molecules that non-specifically bind to the Protein A/Protein G and/or the beads themselves

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

If you are using reducing agents in the sample buffer before gel loading, try incubating the beads in a sample buffer without reducing agents. Reducing agents such as DTT or ß-mercaptoethanol will reduce disulfide bridges and result in release of antibody light and heavy chains. You may also elute the protein by lowering the pH, to leave the antibody bound to the beads.

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The crosslinking is never a 100% efficient. This means that some antibody is not crosslinked and may come off with elution. You can perform a washing step with low pH directly after crosslinking to remove non-crosslinked antibodies. Remember to bring the pH back to normal before performing your immunoprecipitation step.

Alternatively, if reducing agents are included in the buffers, they could dissociate the four chains of the antibody, releasing those which are not covalently attached to the beads.

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The antibody will come off the beads during elution if the antibody is not covalently crosslinked to the beads. Antibodies attach to Protein A or Protein G by non-covalent binding similar to that which occurs between the protein/antigen and the antibody. When you break these non-covalent interactions, antigens and antibodies will both elute.

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The 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.

Find additional tips, troubleshooting help, and resources within our Dynabeads Nucleic Acid Purification Support Center as well as our Protein Immunoprecipitation (IP), Co-Immunoprecipitation (Co-IP), and Pulldown Support Center.

Dynabeads Protein A and Dynabeads Protein G magnetic beads are excellent for immunoprecipitation/co-immunoprecipitation (IP/Co-IP). The advantages are:

- Elimination of background
- Gentle pulldown
- Reduced protocol time


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Due to the very low non-specific binding properties of Dynabeads Protein A- and Dynabeads Protein G-coated magnetic beads, pre-clearing is generally not required with these kits. However, pre-clearing may help if a lot of non-specific protein binding to the beads is observed.

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

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The Wash and Bind buffer is 0.1 M sodium phosphate (pH 8.2), containing 0.01% (v/v) Tween-20.

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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 of the same class or isotype.

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

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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 or 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 Dynabeads Protein G. A larger 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. It is important to keep the Dynabeads Protein G in suspension during this step
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 Dynabeads Protein G-Ig complex can be used for immunoprecipitation.

Ig Elution Procedure

Elution of Igs is, in this example, performed by lowering the pH using 0.1 M citrate buffer (pH 2-3). The acid pH needed depends on the species and Ig subclass, but at pH 3.1 most Igs will elute.

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) dihydrate (MW=178) in distilled water and mix to dissolve. Bring the final volume to 1 L with more 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.

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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. For example, see our cross-linker BS3 (Cat. No. 21580), which we recommend in this case. Note that optimization of the cross-linking protocol may be required.

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Glycerol most likely inhibits non-specific hydrophobic interactions and therefore high concentrations of glycerol might affect the high 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 mixture could be diluted instead by following the protocol, and this dilution may be enough to avoid dialysis. This would need to be tested empirically.

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

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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 nanogram range. In some cases, pictograms of target can be detected.

Find additional tips, troubleshooting help, and resources within our Protein Immunoprecipitation (IP), Co-Immunoprecipitation (Co-IP), and Pulldown Support Center.

Within practical limits, the elution volume can be scaled up or down to suit your experiment. However, 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 with a lot of bound 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.

Find additional tips, troubleshooting help, and resources within our Protein Immunoprecipitation (IP), Co-Immunoprecipitation (Co-IP), and Pulldown Support Center.

There are several methods to quantify the amount of antibody bound to the beads. The crudest method is to measure the concentration of antibody in the coupling reaction before and after antibody attachment. Either fluorescence measurements or absorbance at 280 nm can be used. Alternatively, you could measure the amount of antibody bound to the beads by fluorescence, chemiluminescence, or radiolabeling detection methods.

Find additional tips, troubleshooting help, and resources within our Dynabeads Nucleic Acid Purification Support Center as well as our Protein Immunoprecipitation (IP), Co-Immunoprecipitation (Co-IP), and Pulldown Support Center.

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.

Find additional tips, troubleshooting help, and resources within our Protein Immunoprecipitation (IP), Co-Immunoprecipitation (Co-IP), and Pulldown Support Center.

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.

Find additional tips, troubleshooting help, and resources within our Dynabeads Nucleic Acid Purification Support Center as well as our Protein Immunoprecipitation (IP), Co-Immunoprecipitation (Co-IP), and Pulldown Support Center.

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.

Find additional tips, troubleshooting help, and resources within ourProtein Immunoprecipitation (IP), Co-Immunoprecipitation (Co-IP), and Pulldown Support Center.

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. With EDTA, we highly recommend checking the minimal amount of EDTA that your specific molecules would tolerate for binding to the Dynabeads, and if it will affect your specific application. For some applications, low concentrations of EDTA can be tolerated by Dynabeads. On the other hand, using 10 mM EDTA with heating affects the binding of biotin molecules to Dynabeads streptavidin.

Find additional tips, troubleshooting help, and resources within our Dynabeads Nucleic Acid Purification Support Center as well as our Protein Immunoprecipitation (IP), Co-Immunoprecipitation (Co-IP), and Pulldown Support Center.

Yes, they are compatible with 6-8 M Urea when used during post-coupling steps.

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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.

Find additional tips, troubleshooting help, and resources within our Dynabeads Nucleic Acid Purification Support Center as well as our Protein Immunoprecipitation (IP), Co-Immunoprecipitation (Co-IP), and Pulldown Support Center.

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 (https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-assays-analysis/immunoprecipitation.html#products).

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Protein A, Protein G, and Protein A/G bind almost exclusively to the IgG class of antibodies, but their binding properties differ among species and subclasses of IgG. Protein L binds in the variable fragment of some kappa light chains and can react with any immunoglobulin, not just IgG, as long as the correct kappa light chains are present. Protein L does not bind lambda light chains and certain kappa chains of different species.

-Protein A is generally preferred for rabbit, pig, dog, and cat IgG.
-Protein G has better binding capacity for a broader range of mouse and human IgG subclasses (e.g., IgG1 vs. IgG2)
-Protein A/G is a recombinant fusion protein that includes the IgG-binding domains of both Protein A and Protein G. Therefore, Protein A/G is ideal for binding the broadest range of IgG subclasses from rabbit, mouse, human, and other mammalian samples.
-Protein L binds to certain immunoglobulin kappa light chains. Because kappa light chains occur in members of all classes of immunoglobulin (i.e., IgG, IgM, IgA, IgE and IgD), Protein L can purify these different classes of antibody. However, only those antibodies within each class that possess the appropriate kappa light chains will bind. Generally, empirical testing is required to determine if Protein L is effective for purifying a particular antibody. It binds only Vk1 in mouse and VkI, VkIII and VkIV in human.
Read more about the general characteristics of Ig-binding proteins (https://www.thermofisher.com/us/en/home/life-science/antibodies/antibody-purification-kits-reagents.html) and (https://assets.thermofisher.com/TFS-Assets/LSG/Application-Notes/TR0034-Ab-binding-proteins.pdf).

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