Dynabeads and immunoprecipitation (IP) general advantages

Dr. Ketil W. Pedersen explains the advantages of Invitrogen Dynabeads, and why they are the most used and cited products in the immunoprecipitation (IP) field, in the first of this three-blog series.

Publication trends show that immunoprecipitation (IP) using magnetic beads is the fastest growing method for IP. Invitrogen Dynabeads are the most used and cited products in this field. Why? Because Dynabeads offer a balance of capacity/yield, reproducibility, purity, and cost for capture of specific proteins (immunoprecipitation) and protein complexes (co-immunoprecipitation). Below are general advantages of using Dynabeads magnetic bead technology for immunoprecipitation.

1) Rapid binding kinetics with magnetic bead technology.

Due to the fact that number of magnetic beads per given volume is approximately 1000x higher than for the same volume of Sepharose slurry, the chance for Dynabeads to “hit” the target is far greater than with Sepharose. Learn more about the History of Dynabeads and see how they compare to Sepharose and other magnetic beads technologies.

Uniformity:

Optimal reaction kinetics between the Dynabeads™ and the desired target. This allows for binding of the target and the bead to occur rapidly and efficiently. In many cases, binding is completed in less than 10 min.

Spherical:

The true spherical shape eliminates clumping and non-specific binding associated with irregularly shaped particles.

High Surface Area:

The uniform nonporous surface area provides efficient binding and optimal accessibility of your target probe (e.g., primary antibody, protein).

Dynabeads magnetic beads vs. other magnetic bead-based solutions

Figure 1. The magnetic bead you choose will affect your results. Dynabeads magnetic beads have a defined surface to carry out the necessary binding, with no inner surface to trap unwanted proteins. (A) Dynabeads products are the most uniform, monodispersed superparamagnetic beads, manufactured with highly controlled product quality to help ensure the highest degree of reproducibility. (B–D) Magnetic particles from alternative suppliers have variable shapes and sizes that trap impurities, resulting in lower reproducibility and increased nonspecific binding.

2) Incubation time

Due to the rapid binding kinetics the protocol is usually very short.

3) Background

Due to the rapid binding kinetics and the short incubation time, the background will also be very low.

4) Trapping of impurities

No internal volume for binding and/or trapping of impurities.

5) Dynabeads uniform physical characteristics produce repeatable results

Dynabeads are non-porous uniform, superparamagnetic, monodispersed highly cross-linked polystyrene microspheres consisting of an even dispersion of magnetic material throughout the bead. The magnetic beads are coated with a thin layer of highly cross-linked polystyrene shell which encases the magnetic material and prevents any leakage from the beads or trapping of ligands in the bead interior. This provides 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. Find the right bead by application type using the Dynabeads Selection Guide.

Amount of Antibody (Ab) used: Dynabeads Protein G

Ab binding to the Dynabeads is usually very fast due to rapid binding kinetics. We have performed several experiments demonstrating that increasing the time for incubation of magnetic beads with Ab is not required. Extending the incubation time to 1h or even overnight will not result in much more Ab binding to the beads. Keep in mind that binding efficiency will be dependent on which bead you have and the Ab subclass, e.g. IgG1 will bind efficiently to Protein G but to less extent to Protein A beads. When incubating your Dynabeads coated with Ab with the lysate containing target of interest, you have to optimize that as this will depend on your primary Abs affinity for the Ag. In an experiment targeting CD9 we are getting strong signal on western blot after only 10 min incubation with target (both for manual & automated workflows). See more on Immunoprecipitation protocols using Dynabeads and KingFisher instruments.
The elution efficiency will depend on the volume. Increased volume will result in higher elution efficiency. Our inhouse experiment shows that increasing the elution volume from 20 µL to 100 µL will increase elution efficiency by 20 %. However, our choice of using 20 µL is related to the fact that we would like to add all sample on the gel and the well capacity (BOLT™ gels) is 40 µL/well. In addition, we do get very strong signal after 20 µL elution in our internal system. This needs to be optimized for each immunoprecipitation.
Amount of Primary Ab: We recommend titrating this. In general we recommend between 1-10 µg Ab per 50 µL Dynabeads (per IP). The yield of target will depend on how good your primary Ab is.

6) Reproducible Results using Dynabeads

Reproducibility is due to easier practical handling of magnetic beads, for instance pipetting. No centrifugation steps are required, or pre-clearing.