Dynabeads Magnetic Beads: Tips and Tricks

In the last of this three-blog series, Dr. Ketil Pedersen shares some tips and tricks for using Dynabeads magnetic beads.

Dynabead and Immunoprecipitation – detection limit

When using HRP or radioactivity in combination with a good antibody, very little target is required for detection. More target is required when using an ALP based detection system. How this relates to PG is not straightforward to answer. However, using a sensitive detection system one can most likely detect in the range of NG and possibly PG.

Background on Dynabeads Protein G

Protein G is coated onto hydrophilic beads. If your background is protein-mediated then we typically suggest having a combination of blocking protein and non-ionic detergent both in the coupling and washing buffer to reduce non-specific binding.

Which Dynabead magnet to choose?

The magnetic stand recommended will depend on the sample volume. For an online tool for selecting the right magnet, see the Dynabeads Selection Guide.

up to 2 mL sample volume: DynaMag-2 cat no 12321D
up to 15 mL sample volume: DynaMag-15 cat no 12301D
up to 50 mL sample volume: DynaMag-50 cat no 12302D
pulls beads to the side of each well. Compatible with PCR-strips and 96-well plates (200 µL) non-skirted and half-skirted: DynaMag™-96 Side 123-31D
pulls beads to the bottom of each well. Compatible with PCR-strips and 96-well plates (200 uL) non-skirted and half-skirted: DynaMag™-96 Bottom 123-32D

Dynabeads and their elution conditions

Mild elution methods include change of pH (lowering pH is most frequently used), change of ionic strength (high salt concentration buffers (e.g. NaI, KI, MgCl, KCl) can be used. Please note that the protocols may need to be optimized depending on the antibodie’s affinity towards the target. The coated Dynabeads with antibody may be reused several times using mild elution methods like low pH or high salt concentration. The success of elution depends on affinity of immobilized antibody and the captured target. In addition, the reusability of the immobilized antibody using low pH also depends on sensitivity of the immobilized antibody toward this type of treatment. Using high salt concentration as elution method assures several rounds of reusing beads but using low pH, the yield will normally decrease after couple of rounds of reusing.

Are Dynabeads Protein A and Protein G pre-blocked with BSA?

No. The Dynabeads are coupled with Protein A or Protein G. The hydrophilic surface is blocked with BSA. The Dynabeads™ Protein G Immunoprecipitation Kit includes all reagents and buffers required to perform IP using your own antibody.

How to block non-specific binding to Dynabeads Protein A and G

Use a more stringent washing buffer for washing.
Add a non-ionic detergent (Tween® 20 or Triton® X-100) to the washing buffer, in concentrations between 0.01 – 0.1 %.
If the beads are blocked before precipitation, add identical blocker to the washing buffer.
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.

Dynabeads Protein A and G – Abs coming off despite cross-linked

Cross-linking will never be 100 %. Some antibodies are not cross-linked and may come off under elution.
Perform a washing step with low pH directly after cross-linking to remove non-cross-linked antibodies. Remember to bring the pH back to normal before IP.

Dynabeads Protein A and G – Low binding

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 proposed in the package inserts, increase the amount of beads or the concentration of your antibody during coupling.
Increase the incubation time.
Try another antibody.

Dynabeads Protein A and G – Non-specific binding

Use more stringent washing buffer for washing.
Add a non-ionic detergent (Tween-20 or Triton X-100) to the washing buffer, in concentrations ranging 0.01-0.1%.
If the beads are blocked before precipitation, add identical blocker to the washing buffer.
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.

Dynabeads compatibility with Organic solvents

Two important factors for all solvents are concentration and incubation time. The beads themselves are compatible with several solvents and leave the iron oxide precipitates untouched:

Solvent Compatible with Dynabeads

Ethanol (70 % is used to sterilize the beads/washings),
Methanol,
Acetone,
Isopropanol,
Diglym,
DMSO (dimethyl sulfoxide)
DMF (dimethylformamide).
THF (tetrahydrofuran) is similar to diglym and should work fine with Dynabeads
Acetonitrile
Toluene

Co-Immunoprecipitation (co-IP)

The study of protein-protein interactions often involves very short lived and weak interactions, such as adaptor proteins binding to phosphorylated Tyrosine residues on the cytosolic tail of activated receptors. Or if one wants to detect all molecules binding to a certain protein such as the very interesting study of Praefke et al 2004 who identified binding partners to adaptor protein 2 (AP-2), PIP2 and clathrin (Praefcke et al The EMBO Journal VOL 23 | NO 22 | 2004).

Very interesting work was also done by the group of Albert et al. 2007 (Nature 450, 683-694) who studied nuclear pore complexes and interactions followed by downstream mass spec. This group used the Dynabeads M-270 Epoxy in combination with optimized buffers as these beads have very low background to avoid contaminate the preparation. They did spend a significant amount of time to find the correct buffer composition to capture these weak interactions. These buffers are part of our Dynabeads Co-Immunoprecipitation Kit which includes Dynabeads M-270 Epoxy and Ab coupling buffers in addition to co-IP buffers. These buffers offer the opportunity to optimize and fine tune weak and transient interactions between short amino acid sequences in different proteins in terms of stringency.

Components that can be varied to increase or decrease stringency of the Co-IP

Salts
Detergents
Dithiothreitol (DTT)
Protease Inhibitors

Buffer modifiers

Sodium Chloride (e.g., 1 M NaCl)
Dithiothreitol (e.g., 1 M DTT)
Magnesium Chloride (e.g., 1 M MgCl2)
Potassium Acetate (e.g., 1 M KOAc)
Tween™-20
Triton X-100
Protease inhibitors without EDTA

If already optimized the buffer it is also possible to buy the Dynabeads Antibody Coupling Kit which contains the Dynabeads M-270 Epoxy and Ab coupling buffers

In terms of Ab coupling to the beads, coupling of large amounts of Abs (20-30 µg Ab/mg beads) is not recommended. If adding 20 µg of Abs to the beads the total amount of antibodies covalently coupled to the beads will not exceed 13 µg/mg beads. Up to 10 µg one will find a linear relationship between added Ab and coupled Ab. Above these concentrations the Abs will precipitate down to the bead surface, but covalent bonds will be reduced. Actually, less than 10 µg Ab ensures efficient covalent binding (no leakage) and top functionality.