Dynabeads® His-Tag Isolation & Pulldown -"DISCONTINUED" - FAQs

When stored in unopened vials at 2-8 degrees C, Dynabeads magnetic beads are stable until the expiration date printed on the label. Freezing or drying of the Dynabeads magnetic beads is not recommended. Dynabeads magnetic beads should be fully resuspended before use.

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Yes. The Dynabeads magnetic beads provide an excellent solid-phase for electron microscopy of the attached organelles/compartments. The beads can be sliced with a diamond knife and viewed under electron microscopy.

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Organelles separated with the Dynabeads magnetic beads immunomagnetic method can be assayed by all conventional techniques, such as chemicals, radiolabelling, enzyme immunoassays or SDS-PAGE analysis. The bead surface provides gentle handling so that organelles typically retain their in vivo functions after isolation. The high yield is due to no physical stress or loss of sample since no cumbersome centrifugations or washing steps are necessary.

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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.

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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 using 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.

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It is very important to properly homogenize the sample before use. Homogenization may be easier for some samples such as liver, than for other samples such as yeast. If nuclei are broken during homogenization, DNA will be released and may cause clumping of organelles. Clumping of organelles in the unpurified cellular material will make the immuno-isolation technique difficult

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The capacity of the Dynabeads magnetic beads depends on the type of organelle isolated, the primary antibody/ligand chosen, as well as the type of starting material. The efficiency of the Dynabeads magnetic beads immuno-isolation technique can also be correlated with the density of specific antibody on the bead surface.

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It is essential to identify an appropriate antigen and use a high-affinity antibody that recognizes the antigen in its native, non-denatured state. The epitope on the subcellular fraction must be free to bind the specific antibody. If the epitope is “buried” within the fraction and not exposed on the surface, it may not be possible to isolate the target organelles.

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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.

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Immunomagnetic separation enables specific purification of compartments regardless of their physical properties. As opposed to standard density gradient centrifugations, immunomagnetic techniques enable separation of different compartments of equal density as well as co-isolation of immunogenically equal compartments of differing densities. The fast and easy protocol for isolation reduces time and labor, and the result is pure fractions available for further analyses.

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Which product to choose depends on your ligand, target, sample and application. Both 1 micron (MyOne), 2.8 micron (M-270/M-280) and 4.5 micron (M-450/M-500) Dynabeads magnetic beads can be used. If the sample is very viscous, 4.5 micron beads are recommended. If the target is very fragile, smaller beads are recommended, as they will move more slowly towards the magnet, reducing the risk of squeezing the target. Below is a short product guide showing which beads can be used to immobilize different ligands:

For antibody ligands, use anti-mouse, anti-rat or anti-rabbit coated beads or Dynabeads magnetic beads Protein A/G (2.8 micron beads), Dynabeads magnetic beads M-280 Tosylactivated, Dynabeads magnetic beads M-450 Epoxy or Dynabeads magnetic beads M-500 Subcellular.

For biotinylated ligands, use Dynabeads magnetic beads M-280 Streptavidin. The hydrophilic, negatively charged Dynabeads magnetic beads M-270 Streptavidin or Dynabeads magnetic beads MyOne Streptavidin C1 may also be used.

For protein/peptide ligands (other than antibodies), use Dynabeads magnetic beads M-270 Epoxy, Dynabeads magnetic beads M-270 Amine, Dynabeads magnetic beads M-450 Epoxy.

For histidine affinity-tagged ligands, use Dynabeads magnetic beads His-Tag Isolation and Pulldown beads (1 micron size).

Find more information about Dynabeads magnetic beads at http://www.thermofisher.com/Dynabeads magnetic beads.

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You can choose from several types of Dynabeads magnetic beads for coating with antibody against the subcellular component of choice. We currently do not offer any ready-to-use Dynabeads magnetic beads against a particular subcellular component, and recommend that you choose the beads that work best with your antibody and your application.

The following groups of Dynabeads magnetic beads can be used for this purpose:

(1) Surface Activated Dynabeads magnetic beads
Antibodies from any origin can be conjugated directly to these uncoated beads through covalent bonds. For better orientation of primary antibody on the beads, a secondary antibody (linker antibody) can be coated on the beads first. The Surface Activated Dynabeads magnetic beads are provided in different sizes (4.5, 2.8 and 1 micron) and the larger ones are mostly preferred for this application.

(2) Secondary Coated Dynabeads magnetic beads
These Dynabeads magnetic beads are pre-coated with a secondary antibody and depending on their specificity, they can be easily coated with your primary antibody within a short time period. These beads are provided in two sizes, 4.5 and 2.8 micron. In addition to these beads, our Dynabeads magnetic beads Protein A or Protein G can also be easily coated with antibody from several species. Please see the following links for information about all available Secondary coated Dynabeads magnetic beads and Dynabeads magnetic beads Protein A and Protein G.

(3) Streptavidin Coated Dynabeads magnetic beads
These Dynabeads magnetic beads are coated with streptavidin and require a biotinylated antibody. These beads are provided in two different sizes, 2.8 and 1 micron.

Find more information about Dynabeads magnetic beads at http://www.thermofisher.com/Dynabeads magnetic beads.

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There are several ways of preventing non-specific binding.

Increased number of washing steps, washing time and volume often helps.

Adjusting pH can also help.

Higher salt concentrations almost always help; up to 500 mM NaCl may be used.

Other recommended additives for binding/washing buffer are non-ionic detergents such as Tween 20 or NP-40.

Glycerol can be added to prevent hydrophobic interactions and sometimes ethanol can do the same.

Using fewer beads may sometimes give better results, and reduced incubation time for binding might help too.

Finally, to avoid non-specific binding to the beads it is important to choose the right Dynabeads magnetic beads product for your specific application.

For Dynabeads magnetic beads His-Tag Isolation & Pulldown, low concentrations of imidazole (5-20 mM) may be added to both binding and washing buffers to reduce non-specific binding.

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This new Dynabeads magnetic beads product replaces Dynabeads magnetic beads TALON. Its surface chemistry is cobalt-based, which offers exceptionally fast binding kinetics and a high yield per microliter of beads.

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