Dynabeads™ Protein G for Immunoprecipitation, 1 mL - FAQs

Please review the following possibilities for why your Dynabeads magnetic beads are not pelleting:

- The solution is too viscous.
- The beads have formed aggregates because of protein-protein interaction.

Try these suggestions: - Increase separation time (leave tub on magnet for 2-5 minutes)
- Add DNase I to the lysate (~0.01 mg/mL)
- Increase the Tween 20 concentration to ~0.05% of the binding and/or washing buffer.
- Add up to 20 mM beta-merecaptoethanol to the binding and/or wash buffers.

Find additional tips, troubleshooting help, and resources within our Dynabeads Nucleic Acid Purification Support Center.

For biotin-labled DNA that is less than 1 kb, we recommend you use Dynabeads M270 Streptavidin and MyOne C1 magnetic beads. We recommend our Dynabeads KilobaseBINDER Kit, which is designed to immobilize long (>1 kb) double-stranded DNA molecules. The KilobaseBINDER reagent consists of M-280 Streptavidin-coupled Dynabeads magnetic beads along with a patented immobilization activator in the binding solution to bind to long, biotinylated DNA molecules for isolation. Please see the following link (https://www.thermofisher.com/us/en/home/life-science/dna-rna-purification-analysis/napamisc/capture-of-biotinylated-targets/immobilisation-of-long-biotinylated-dna-fragments.html) for more information in regards to long biotinylated DNA fragment isolation.

Find additional tips, troubleshooting help, and resources within our Dynabeads Nucleic Acid Purification Support Center.

Yes, Dynabeads magnetic beads can be used to isolate single-stranded DNA. Streptavidin Dynabeads magnetic beads can be used to target biotinylated DNA fragments, followed by denaturation of the double-stranded DNA and removal of the non-biotinylated strand. The streptavidin-coupled Dynabeads magnetic beads will not inhibit any enzymatic activity. This enables further handling and manipulation of the bead-bound DNA directly on the solid phase. Please see the following link (https://www.thermofisher.com/us/en/home/life-science/dna-rna-purification-analysis/napamisc/capture-of-biotinylated-targets/preparing-single-stranded-dna-templates.html) for more information in regards to single-stranded DNA capture.

Find additional tips, troubleshooting help, and resources within our Dynabeads Nucleic Acid Purification Support Center.

Magnetic susceptibility is a measure of how quickly the beads will migrate to the magnet. This will depend on the iron content and the character of the iron oxide. The magnetic susceptibility given for the Dynabeads magnetic beads is the mass susceptibility, given either as cgs units/g or m^3/kg (the latter being an SI unit). For ferri- and ferromagnetic substances, the magnetic mass susceptibility is dependent upon the magnetic field strength (H), as the magnetization of such substances is not a linear function of H but approaches a saturation value with increasing field. For that reason, the magnetic mass susceptibility of the Dynabeads magnetic beads is determined by a standardized procedure under fixed conditions. The magnetic mass susceptibility given in our catalog is thus the SI unit. Conversion from Gaussian (cgs, emu) units into SI units for magnetic mass susceptibility is achieved by multiplying the Gaussian factor (emu/g or cgs/g) by 4 pi x 10^-3. The resulting unit is also called the rationalized magnetic mass susceptibility, which should be distinguished from the (SI) dimensionless magnetic susceptibility unit. In general, magnetic mass susceptibility is a measure of the force (Fz) influencing an object positioned in a nonhomogenous magnetic field. The magnetic mass susceptibility of the Dynabeads magnetic beads is measured by weighing a sample, and then subjecting the sample to a magnetic field of known strength. The weight (F1) is then measured, and compared to the weight of the sample when the magnetic field is turned off (F0). The susceptibility is then calculated as K x 10^-3 = [(F1-F0) x m x 0.335 x 10^6], where K is the mass susceptibility of the sample of mass m. The susceptibility is then converted to SI units.

Find additional tips, troubleshooting help, and resources within our Dynabeads Nucleic Acid Purification Support Center.

There are different methods to check binding of ligands to the beads, including optical density (OD) measurement, fluorescent labeling, and radioactive labeling.

For OD measurement, you would measure the OD of the ligand before immobilization to the beads and compare it with the ligand concentration that is left in the supernatant after coating. This gives a crude measurement of how much protein has bound to the beads.

Protocol:

1.Set spectrophotometer to the right wavelength. As a blank, use the Coupling Buffer.
2.Measure the absorbance of the Pre-Coupling Solution. A further dilution may be necessary to read the absorbance, depending upon the amount of ligand added.
3.Measure the absorbance of the Post-Coupling Solution. A dilution may be necessary to read the absorbance.
4.Calculate the coupling efficiency, expressed as the % protein uptake, as follows. [(Pre-Coupling Solution x D) - (Post-Coupling Solution x D)] x 100/(Pre-Coupling Solution x D) where D = dilution factor.

For fluorescent labeling, we suggest negatively quantifying the amount of ligand bound by measuring ligand remaining in the coupling supernatant (compared to the original sample), rather than directly measuring the ligands on the beads. Add labeled ligand to the beads, and measure how much ligand is left in the supernatant (not bound to the beads). By comparing this with the total amount added in the first place, you can then calculate how much of the ligand that has been bound to the beads. Keep in mind that the Dynabeads magnetic beads are also autofluorescent, which is why direct measuring of fluorescence of the bead-bound ligands is not recommended, but rather this indirect approach. The label could be, for example, FITC/PE. Some researchers perform a direct approach with success (using a flow cytometer).

Radioactive labeling is the most sensitive method of the three, but it is also the most difficult one. It involves radioactively labeling a portion of the ligand. We use radiolabeled I-125 in tracer amounts and mix it with "cold" ligands in a known ratio before coupling. The absolute quantities for the ligand on the beads should be obtained by measuring the beads in a scintillation (gamma) counter and comparing the cpm with a standard.

Protocol:

1.Take out an appropriate amount of beads and wash the beads in 1 mL of binding buffer.
2.Pipette out desired amount of human IgG in a separate tube.
3.Mix the human IgG with I-125-labeled human IgG (30,000 - 100,000 cpm).
4.Dilute the mixture of human IgG and I-125-labeled human IgG to 100 mL in binding buffer.
5.Incubate for 30 minutes at room temperature and measure the cpm in a scintillation counter.
6.Wash the beads (with coating) four times, and measure cpm again.
The % binding is calculated by using the equation : (cpm after washing/cpm before washing)x100%.

Find additional tips, troubleshooting help, and resources within our Dynabeads Nucleic Acid Purification Support Center.

Dynabeads magnetic beads come in three sizes: 4.5 µm (M-450), 2.8 µm (M-270/M-280), and 1 µm (MyOne beads). The largest of the Dynabeads magnetic beads is ideal for big targets like cells. The 2.8 µm beads are recommended for proteomics and molecular applications. The smallest of the beads, 1 µm, are ideal for automated handling.

Find additional tips, troubleshooting help, and resources within our Dynabeads Nucleic Acid Purification Support Center.

In general, short sonication is a good way to reduce aggregation of the beads and ensure optimal homogenous conditions at the time of ligand addition when coating the beads. When target is bound to the beads, more care is needed, as the binding might break. The streptavidin beads themselves should tolerate sonication. We have not tested sonication for long periods, but 5 minutes is fine. We do not have information about the streptavidin-biotin interaction being broken by such treatment.

Find additional tips, troubleshooting help, and resources within our Dynabeads Nucleic Acid Purification Support Center.

If desired, the uncoated epoxy or tosylactivated beads can be sterilized by washing with 70% ethanol. Coated beads cannot be sterilized.

Find additional tips, troubleshooting help, and resources within our Dynabeads Nucleic Acid Purification Support Center.

Dynabeads magnetic beads are uniform, non-porous, superparamagnetic, monodispersed and highly cross-linked 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).

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 magnetic beads and Dynabeads Protein G magnetic beads are excellent for co-IP/IP.

The advantages are:
(1) Elimination of background
(2) Gentle pulldown
(3) Reduce protocol time

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

Using Dynabeads Protein G magnetic beads or Dynabeads Protein A magnetic beads by themselves 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 magnetic beads or Dynabeads Protein A magnetic beads bound to an irrelevant IgG.

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

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 non-specific binding by performing more stringent washing and adding Tween 20 detergent (concentrations of 0.01-0.1%) to the washing buffer.

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

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

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

To avoid non-specific binding to your Dynabeads magnetic beads, try the following suggestions:
-Use a more stringent washing buffer for washing.
-Add a non-ionic detergent (Tween 20 detergent or Triton X-100 detergent) to the washing buffer, in concentrations between 0.01-0.1%.
-Increase the number of washing steps.
-Prolong the washing steps.
-Decrease incubation time (beads and sample).
-Try the indirect method.
-Decrease the antibody concentration.
-A pre-clearing step may be performed to remove molecules that non-specifically bind to the Protein A/Protein G or the beads themselves.

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

If you get low binding to your Dynabeads magnetic beads, try the following suggestions
-Verify 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 method, try the direct method. Conversely, if you have used the direct method, try the indirect method.
-Check the amount of beads and sample volume. With reference to the capacity of different beads stated in the package inserts, increase the amount of beads or the concentration of your antibody during coupling.
-Increase the incubation time.
-Try another antibody.

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

As a starting point, use 5 µg antibody per reaction (50 µL Dynabeads magnetic beads). The amount of antibody needed depends on the abundance of target antigen, but we have used 5 µg when doing IP of HSA from diluted serum samples.

50 µL Dynabeads magnetic beads per reaction is suitable, but again this will depend on sample volume and whether it is a qualitative or quantitative assay. If the amount of antigen in the sample is small, you may try to increase incubation time with the IgG-coupled beads and sample (i.e., to 1 h or more).

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 beta-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, which will leave the antibody bound to the beads.

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

The process of cross-linking is not 100% efficient, therefore, the antibodies that are not cross-linked may come off during elution.

To minimize this problem, perform a washing step at low pH immediately after cross-linking to remove non-cross-linked antibodies. Remember to bring the pH back to normal before immunoprecipitation.

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

If your beads have lost function after cross-linking, you can try the following suggestions:

Try immunoprecipitation without cross-linking the antibody to the beads.

Try a different cross-linker.

To prevent co-elution of the antibody, try one of our surface-activated Dynabeads products. This allows you to conjugate the antibody to the beads directly, through covalent binding.

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

The antibody will come off the beads during elution if the antibody is not cross-linked to the beads. The antibody is attached to Protein A or Protein G through affinity binding, like the protein is attached to the antibody through affinity binding. When you break one of the affinity bonds, you necessarily will break the other affinity bonds as well.

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

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.

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

Dynabeads Protein A and G magnetic beads are supplied at a 30 milligram per milliliter concentration. The beads themselves will be at a concentration of 1.8-2.1 x 10⁹ beads per milliliter.

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

Please follow these suggestions if Protein G or Protein A comes off the Dynabeads:

Alternative (1): Wash the beads prior to use in 100 mM glycine, pH 11.3, followed by wash in 200 mM glycine, pH 2.8, for a very short period of time. Immediately transfer the beads to PBS with 0.01% Tween 20 detergent.

Alternative (2): Do the elution step in glycine.

Alternative (3): Cross-link the antibody to the beads in order to covalently attach them to the beads, which will also cross-link Protein G.

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

Dynabeads Protein G magnetic beads have a binding capacity of approximately 8 µg human IgG per mg beads. The amount of antibody captured on the beads depends on the affinity of the Protein G to the specific antibody, concentrations of both antibody and Dynabeads Protein G magnetic beads in the starting sample, volumes and binding conditions (pH, salt, etc.) and incubation time. The highest binding capacity noted for these beads under optimal conditions was 640 µg mouse IgG/mL beads; e.g., 100 µL of Dynabeads Protein G magnetic beads will allow isolation of 25 µg mouse IgG1 (about the same affinity as human IgG1, IgG2, IgG3, and IgG4) from a 600 µL sample containing 100 µg per mL mouse IgG1. In this sample, there is a total of 60 µg of mouse IgG1 and immobilization of 25 µg is almost 50%. To ensure the best possible binding in each case, there are a few important factors to consider that influence the binding: the concentration of antibody and beads (keep both high) and the pH of the coupling reaction (which should be kept between 5 and 7). When the total amount of antibody is kept constant but the volume of the reaction is reduced 10 times (from 500 µL to 50 µL), the binding of the antibody is about 30% better. Due to rapid binding kinetics, the maximum amount of Ig binding is obtained after 10-40 minutes of incubation.

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

Dynabeads Protein G magnetic beads and Dynabeads Protein A magnetic beads have Protein G or Protein A respectively, coated onto hydrophilic beads. To reduce background due to non-specific binding, we recommend adding a non-ionic detergent (Tween-20 or Triton X-100) to the washing buffer, in concentrations between 0.01-0.1%.

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

Dynabeads Protein A magnetic beads and Dynabeads Protein G magnetic beads are perfect for small-scale IgG purification and immunoprecipitation. They have the following advantages:

They require no columns, centrifugations, or time-consuming pretreatment of your samples.

They ensure easy handling and a simple protocol (workflow).

They are gentle, placing minimal physical stress on precious proteins.

The starting sample can be saliva, plasma, ascites, serum, tissue cultures, or hybridoma supernatants.

The recombinant protein A or G on the beads contains no albumin binding sites, thus avoiding co-purification of contaminating proteins.

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

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, Dynabeads Co-Immunoprecipitation Kit (Cat. No. 143-21D, Dynabeads M-270 Epoxy magnetic beads, buffers for covalent coupling, and buffers optimized for co-immunoprecipitation; see Alber F et al. (2007) Determining the architectures of macromolecular assemblies. Nature 450, 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.

Thus, there are several products that can be considered for performing immunoprecipitation depending on preferences. If the target is the same as heavy or light chain antibody, we recommend covalently binding the antibody to the bead surface. This can be done either by cross-linking the antibody to beads coated with Protein A or G or with secondary antibody or by using one of the surface-activated Dynabeads products. As your primary antibody can be used in combination with Dynabeads Protein G magnetic beads (Cat. No. 100-03D) or Immunoprecipitation Kit - Dynabeads Protein G magnetic beads (Cat. No. 100-07D, contains immunoprecipitation buffers as well) or Dynabeads Sheep Anti-Mouse IgG magnetic beads, using a cross-linker will ensure covalent binding of your primary antibody. Depending on the antibody, the functionality of the antibody can be affected. The other option would be the surface-activated Dynabeads products. This is an easy but more time-consuming approach, since the coupling takes an overnight incubation, but it ensures functional antibodies that are not eluted off during elution. For this approach, we recommend the Dynabeads Antibody Coupling Kit (Cat. No. 14311D, contains both surface-activated beads and optimized buffers for covalent coupling).

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.

Storage should be at 2-8 degrees C. Freezing Dynabeads magnetic beads is not recommended. Provided the Dynabeads magnetic beads are stored correctly, quality is guaranteed until the expiration date stated on the label.

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 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 that 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.

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

Standard elution methods are used to dissociate the isolated protein from Dynabeads magnetic beads. The most suitable elution method depends on the characteristics of the isolated protein and the desired downstream application of the eluted protein. In many cases, Dynabeads magnetic beads can be recovered for reuse after elution. Some standard elution methods are listed below.

-pH change: elution can be achieved by reducing pH (for example, by using 0.1 M citrate (pH 2-3) as the elution buffer). Dynabeads magnetic beads are stable between pH 4-13. If the Dynabeads magnetic beads are exposed for a prolonged period of time to pH below 4, the beads may be adversely affected.

-Change of ionic strength: high-salt concentration buffers (e.g., NaI, KI, MgCl, KCl) can be used to elute isolated proteins. Optimization is required, by step-wise elution starting at 1 M and increasing to 3 M.

-Affinity elution: with this method, the eluting agent competes for the binding of the protein or the binding of the ligand e.g., elution of glycoproteins from a lectin coupled to Dynabeads magnetic beads may be achieved by the addition of the free sugar.

-Denaturing eluents: as a last resort, denaturing eluents such as chaotropic salts may be used to alter the structure of the protein. The proteins on the bead surface and the eluted proteins will be denatured.

-Polarity reducing agents : substances that reduce the polarity of the buffer often disrupt the hydrophobic interactions between antibody and protein. Dioxane or ethylene glycol may be used to reduce the polarity of the eluent.

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.

Superparamagnetic means that the Dynabeads magnetic beads exhibit magnetic properties when placed within a magnetic field, but have no residual magnetism when removed from the magnetic field.

This means that your targeted cells, proteins, or nucleic acids are only subjected to magnetic forces during the time the beads are on the magnet. The beads do not aggregate, but remain evenly dispersed in suspension.

Find additional tips, troubleshooting help, and resources within our Dynabeads Cell Isolation and Expansion Support Center.

Yes. The antibodies are covalently bound and should be very stable in your applications.

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

Depending on the antibody coated on the Dynabeads magnetic beads, the shelf life can vary from 24-36 months.
Some kits may have 18 months shelf life depending on other components supplied in the kit. The kits are guaranteed for 6 months from when you receive them.

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.

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


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

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

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

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.

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

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

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

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.

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

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.

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

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.

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

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.

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

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.

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

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


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

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.

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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.

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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.

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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.

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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.

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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.

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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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This depends on the host of the antibodies you are going to use. Protein A and Protein G differ in their binding strength to antibodies/immunoglobulins (Ig) from different species and subclasses.

- Human IgG1, 2, 4: Strong affinity for Protein A; Strong affinty for Protein G
- Human IgG3: No binding to Protein A; Strong affinity for Protein G
- Human IgA, D, E, M: Weak affinity for Protein A; No binding to Protein G
- Mouse IgG1: Weak affinity for Protein A; Strong affinity for Protein G
- Mouse igG2a, 2b, 3: Strong affinity for Protein A; Strong affinty for Protein
- Mouse IgM: Weak affinity for Protein A; No binding to Protein G
- Rat IgG1: Weak affinity for Protein A; Weak affinity for Protein G
- Rat IgG2a: No binding to Protein A; Strong affinity for Protein G
- Rat IgG2b: No binding to Protein A; Weak affinity for Protein G
- Rat IgG2c: Strong affinity for Protein A; Strong affinity for Protein G
- Bovine IgG1: Weak affinity for Protein A; Strong affinity for Protein G
- Bovine IgG2: Strong affinity for Protein A; Strong affinity for Protein G
- Chicken IgY: No binding to Protein A; No binding to Protein G
- Dog IgG: Strong affinity for Protein A; Weak affinity for Protein G
- Goat IgG1: Weak affinity for Protein A; Strong affinity for Protein G
- Goat IgG2: Strong affinity for Protein A; Strong affinity for Protein G
- Guinea pig IgG: Strong affinity for Protein A; Weak affinity for Protein G
- Hamster: Weak affinity for Protein A
- Horse IgG: Weak affinity for Protein A; Strong affinity for Protein G
- Monkey IgG: Strong affinity for Protein A; Strong affinity for Protein G
- Porcine IgG: Strong affinity for Protein A; Strong affinity for Protein G
- Rabbit IgG: Strong affinity for Protein A; Strong affinity for Protein G
- Sheep IgG1: Weak affinity for Protein A; Strong affinity for Protein G
- Sheep igG2: Strong affinity for Protein A; Strong affinity for Protein G

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