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What should I do when the antibodies coupled to Dynabeads Protein A or G magnetic beads have lost function after cross-linking? Product FAQ

Answer

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.

Answer Id:: E6022

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What reactive groups do you offer for your surface-activated Dynabeads magnetic beads? Product FAQ

Answer

We offer tosyl-, epoxy-, carboxylic acid-, and amine-activated Dynabeads magnetic beads. See our bead comparison on the following link (https://tools.thermofisher.com/content/sfs/brochures/Surface_Activated_Dynabeads.PDF).

Answer Id:: E13039

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What reactive groups do you offer for your surface-activated Dynabeads magnetic beads? Product FAQ

Answer

We offer tosyl, epoxy, carboxylic acid and amine activated Dynabeads magnetic beads. Please see the link (https://tools.thermofisher.com/content/sfs/brochures/Surface_Activated_Dynabeads.PDF) for a comparison of the beads.

Answer Id:: E7911

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How are Dynabeads magnetic beads used to isolate a specific target? Product FAQ

Answer

You simply need an antibody or other ligand specific for your target, a magnet, buffers, and solutions. Several antibodies that recognize antigenic sites on organelles are commercially available. You can choose between two techniques:

Direct technique: coat the Dynabeads magnetic beads with your antibody/ligand by incubating the two together for a short incubation period. The coated beads are then mixed with your crude fraction. Target organelles bound to the Dynabeads are separated by the use of a magnet and washing is performed in an equally fast and easy manner.

Indirect technique: mix the antibody/ligand with the target containing crude fraction and incubate to let them bind. Add the Dynabeads magnetic beads to the sample, incubate for a short period to let the target-antibody/ligand complex bind. Separate and wash using a magnet. The indirect technique cannot be used if the target specific antibody/ligand is to be coated directly onto one of the surface-activated beads.

Due to speed and efficiency the direct technique is recommended. However, if the antigen on the target organelles is difficult to access (e.g., buried within the vesicle), the indirect technique may be advantageous. Organelle-specific proteins can be further fractionated using one of the immunomagnetic techniques described.

Answer Id:: E4851

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If Thermo Fisher Scientific does not have a primary-coated Dynabeads magnetic beads product for isolating my target cell type, which alternative Dynabead magnetic beads product can I use instead? Product FAQ

Answer

You may use one of our secondary-coated, surface-activated, or streptavidin-coated Dynabeads magnetic beads, and coat it with a primary antibody to target your cell type.

The Dynabeads magnetic beads product you choose will depend on the primary antibody available for cell targeting and the downstream application for the isolated cells:
-For primary antibodies made in mouse, use the CELLection magnetic beads Pan Mouse IgG Kit, Dynabeads magnetic beads Goat Anti-Mouse IgG, Dynabeads magnetic beads Pan Mouse IgG, Dynabeads magnetic beads Rat Anti-Mouse IgM, Dynabeads magnetic beads Rat Anti-Mouse IgM, or Dynabeads magnetic beads Sheep-Anti Mouse IgG

-For primary antibodies made in rat, use the Dynabeads magnetic beads Sheep Anti-Rat IgG

-For primary antibodies made in rabbit, use the Dynabeads magnetic beads M-280 Sheep Anti-Rabbit IgG

-For primary antibodies made in any species, use the CELLection magnetic beads Biotin Binder Kit, Dynabeads magnetic beads Biotin Binder, Dynabeads magnetic beads FlowComp Flexi, Dynabeads magnetic beads M-450 Epoxy, or Dynabeads magnetic beads M-450 Tosylactivated

Answer Id:: E4670

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How can I isolate organelles using Dynabeads magnetic beads? Product FAQ

Answer

In general, all membrane-bound organelles can be isolated using Dynabeads magnetic beads. In combination with your own organelle-specific antibody, the immunomagnetic isolation approach has several advantages:
The method is rapid and gentle.
It does not require any labor-intensive density gradient or ultracentrifugation.
Organelle populations of different density can be isolated.
Highly pure organelles are obtained.

Isolation techniques:
Several antibodies that recognize antigenic sites on organelles are commercially available. You can choose between two techniques:
(1) Direct technique: coat the Dynabeads magnetic beads with your antibody or ligand by incubating the two together for a short incubation period. The coated beads are then mixed with your crude fraction. Target organelles bound to the Dynabeads magnetic beads are separated using a magnet, and washing is performed in an equally fast and easy manner.
(2) Indirect technique: mix the antibody or ligand with the crude fraction containing the target and incubate to let them bind. Then add the Dynabeads magnetic beads to the sample and incubate for a short period to allow formation of a complex between the target and the antibody or ligand. Separate and wash using a magnet. The indirect technique cannot be used if the target specific antibody or ligand is to be coated directly onto the surface-activated beads.

Due to the speed and efficiency of isolation, the direct technique is recommended. However, if the antigen on the target organelles is inaccessible (e.g. buried within the vesicle), the indirect technique may be advantageous.

Answer Id:: E6146

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How are the proteins coupled to the different Surface-Activated Dynabeads magnetic beads? Product FAQ

Answer

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

Answer Id:: E4720

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How are proteins coupled to surface-activated Dynabeads magnetic beads? Product FAQ

Answer

Dynabeads M-280 Tosyl activated magnetic beads allow direct covalent binding to primary amino or sulfhydryl groups in proteins and peptides at high pH and high temperature.
-Dynabeads M-270 Epoxy magnetic beads allow direct covalent binding to primary amino and sulfhydryl groups in proteins and peptides at neutral pH and across a wide temperature range.
-Dynabeads M-270 Amine magnetic beads allow direct covalent binding through reductive amination of aldehydes, or the use of bifunctional amine-reactive crosslinkers.
-Dynabeads M-270 and MyOne Carboxylic Acid magnetic beads allow covalent amide formation with primary amino groups in proteins and peptides.

Answer Id:: E13036

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What choice of Dynabeads magnetic beads do you offer for isolating organelles if I would like to covalently link my own antibody to the beads? Product FAQ

Answer

We have several surface-activated Dynabeads magnetic beads that allow you to couple your own antibody covalently to the beads. These products include M-450 Tosylactivated beads (Cat. No. 14013), M-280 Tosylactivated beads (Cat. Nos. 14203 and 14204), M-450 Epoxy beads (Cat. No. 14011), M-270 Epoxy beads (Cat. No. 14301), M-270 amine beads (Cat. No. 14308), and M-270 carboxylic acid (Cat. No. 14306D).

Answer Id:: E12139

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I was able to immunoprecipitate my protein using Dynabeads magnetic beads before, but after cross-linking the antibody to the beads I get no protein bands on my gel. Product FAQ

Answer

The binding sites of your antibody might be affected by the cross-linker and your antibody does not recognize the antigen anymore.
-Try IP without cross-linking the antibody to the beads.
-Try a different cross-linker.
-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.

Answer Id:: E5034

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Can Dynabeads magnetic beads be used for phage display and solid-phase biopanning? Product FAQ

Answer

Dynabeads magnetic beads are the ideal solid support for biopanning techniques such as phage display (Eur J Biochem 268:4269-4277) and SELEX. They have been used to identify targets for therapy (J Virol 76(8):3688-3696), diagnostic markers (Nature Biotech 17:67-72) and to generate lead molecules in drug discovery (Cancer Res 62(11):3184-3194).

Biotinylate the target ligand, couple it to Dynabeads Streptavidin magnetic beads and then expose to the phage or random DNA library. You can then separate the positive binders from negatives by magnetic handling. Alternatively, you can immobilize your ligand using Secondary-Coated Dynabeads magnetic beads, Dynabeads Protein A magnetic beads, or Dynabeads Protein G magnetic beads, or a selection of Surface-Activated Dynabeads magnetic beads.

Answer Id:: E6035

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What should I do when the antibodies coupled to Dynabeads Protein A or Protein G magnetic beads have lost function after crosslinking? Product FAQ

Answer

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

Answer Id:: E13065

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I am using surface-activated Dynabeads magnetic beads. When do I need to use a linker secondary antibody? Product FAQ

Answer

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

Answer Id:: E13038

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Which Surface Activated Dynabeads magnetic beads should I choose for immobilizing my protein? Product FAQ

Answer

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

The most frequently used surface activated Dynabeads magnetic beads for protein isolation are the Dynabeads magnetic beads M-280 Tosylactivated. Dynabeads magnetic beads M-280 Tosylactivated are hydrophobic, while the other surface activated beads (Dynabeads magnetic beads M-270 Epoxy, Dynabeads magnetic beads M-270 Amine and Dynabeads magnetic beads M-270 Carboxylic acid and Dynabeads magnetic beads MyOne Carboxylic acid) are all hydrophilic. Dynabeads magnetic beads M-280 Tosylactivated are easy to handle and ideal for covalent binding of antibodies for immunoprecipitation. Other ligands could also be covalently bound to these beads, but they have to tolerate conditions like neutral to high pH and high temperatures (required for covalent bond formation).

Dynabeads magnetic beads M-270 Epoxy is used when the ligand to be immobilized needs to be treated gently and will not tolerate harsh binding conditions like high temperature or pH. Proteins/peptides (other than antibodies) and enzymes are often coupled onto these beads.

Dynabeads magnetic beads M-270 Amine is often used in combination with cross-linkers to create specific surface groups on the beads. Hetro-bifunctional cross-linkers with an amine binding NHS group at one end and another chemical group of your choice at the other end are most frequently used. For example, Dynabeads magnetic beads M-270 Amine can be coated with a hetero-bifunctional cross-linker containing a NHS group at one end and maleimide at the other end to create Dynabeads magnetic beads with a maleimide surface. Since maleimide reacts specifically with sulfhydryl groups, these modified Dynabeads magnetic beads can be used for applications where binding of sulfhydryl groups are desired. Dynabeads magnetic beads M-270 Amine may also be used for direct ligand coupling via aldehyde or ketone groups by Schiff base (imine) formation and reductive amination. In addition carboxylic acid groups on a ligand can be activated with cabodiimide, which results in a direct amide bond formation between the beads and the ligand. Alternatively, a cross-linker may be introduced to the ligand. After activation of the ligand with cross-linker, the free end on the cross-linker has to be amine reactive.

Dynabeads magnetic beads M-270 Carboxylic Acid and Dynabeads magnetic beads MyOne Carboxylic Acid can also be used for immobilizing proteins. The carboxylic acid groups on these beads need to be activated (with carbodiimide) before coupling. The negative surface of these beads may attract positively charged proteins and cause nonspecific binding. This needs to be considered if the these beads are going to be used for immunoprecipitation.

Answer Id:: E4718

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How can antibodies be coupled to Dynabeads magnetic beads? Product FAQ

Answer

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).

Answer Id:: E6004

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