Product FAQ

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

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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Product FAQ

How can I isolate organelles using Dynabeads magnetic beads?

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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Product FAQ

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.

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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Product FAQ

Do you provide inert Dynabeads magnetic beads?

Answer

All Dynabeads magnetic beads are either coated with a ligand or are surface activated, meaning they have a chemical functionality on their surface.

( 1) The most inert beads are Dynabeads M-270 magnetic beads and Dynabeads MyOne Carboxylic Acid magnetic beads. They need activation (with EDC) to react covalently with molecules. One problem is that these beads may form clumps or aggregate and can be difficult to handle at neutral pH (they are easier to handle at lower pH, about 5-6).

(2) Dynabeads M-270 Epoxy magnetic beads can be inactivated by incubation in aqueous solutions (the reason they are shipped freeze-dried), and these beads will become inert due to hydrolysis when they come in contact with water (the required incubation time is unknown, but fairly long). (Please note that Dynabeads M-450 Epoxy magnetic beads is more stable, as it is based on a more hydrophobic particle). Incubation in Tris buffer may also effectively block the epoxy groups (at least a 4 hours incubation at room temperature). Using Tris will add amine groups, and to reduce this effect, it may be better to incubate the coated beads overnight in ethanolamine (0.1 M at 60 degrees C for 1 h or 37 degrees C overnight). This will also inactivate epoxy groups. In addition, you can accelerate the hydrolysis by incubation in water with a high concentration of NaOH (0.5 M at 60 degrees C for 1 h or 37 degrees C overnight). There are also chemical groups on the bead surface other than the remaining epoxy groups that can cause adsorption of molecules.

(3) Smaller beads (because they are more unstable) act as more inert beads.

(4) Tosylactivated beads (both Dynabeads M-280 Tosylactivated magnetic beads and Dynabeads M-450 Tosylactivated magnetic beads) may be inactivated by blocking the active groups with Tris alone. But the first two approaches are likely to work better.

Answer Id: E6199

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Product FAQ

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

Answer

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

Answer Id: E4862

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Product FAQ

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

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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Product FAQ

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?

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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Product FAQ

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

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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Product Literature

Poster: New optimised one micron magnetic bead platform for fast and efficient development of automated immunoassays

Product FAQ

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

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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Product FAQ

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

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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Product FAQ

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

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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Product FAQ

Can you give me an overview for isolating organelles with Dynabeads magnetic beads?

Answer

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. Then mix your coated beads with your crude fraction. Target organelles bound to the Dynabeads magnetic beads 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 allow them bind. Then add the Dynabeads magnetic beads to the sample, incubate for a short period to allow 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.

Answer Id: E12131

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Product FAQ

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

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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Product FAQ

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

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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