Dynabeads™ Protein A for Immunoprecipitation, 50 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.

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.

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.

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

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.

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

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.

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

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.

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

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.

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

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.

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

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

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

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.

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

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).

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

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).

Find additional tips, troubleshooting help, and resources within our Protein Purification and Isolation Support Center.