Immunoprecipitation using Dynabeads® Protein A or G
Are you still using Sepharose®/Agarose slurry?
Thousands of scientists are moving away from Sepharose® and agarose. They now prefer to use magnetic beads. 
Figure 1: Dynabeads® magnetic separation is the fastest growing method for immunoprecipitation. Graphs show percent increase/decrease of published papers using agarose (green), sepharose (blue) and Dynabeads® (red). Source: Google Scholar, Feb. 2011. Sepharose® is a trademark of GE Healthcare companies.
Immunoprecipitation is going magnetic!
While agarose / sepharose slurry worked great when you wanted to purify large amounts of protein or antibody, it turned out not to be so well suited for the smaller scale isolation of specific proteins and protein complex.
The priority of quantity often shifts to a higher priority of quality and specificity. And for this, magnetic beads are perfectly suited. Not just because they are so easy to use, but because they give the results you want!
In just the last 4 years, Dynabeads® are used and cited in over 4,200 published articles.
The priority of quantity often shifts to a higher priority of quality and specificity. And for this, magnetic beads are perfectly suited. Not just because they are so easy to use, but because they give the results you want!
In just the last 4 years, Dynabeads® are used and cited in over 4,200 published articles.
How to avoid 2 typical problems
What is Most Important for Immunoprecipitation?
| No background Minimal non-specific binding | Signal to Noise Easy and efficient washing | Easy handling Magnetic separation protocol | ||||||
| Ab savings All binding on outer surface | Time and Effort 30 minutes start to finish | Reproducibility Product and handling consistency |
These are the main reasons why scientists now prefer to use Dynabeads® for their immunoprecipitaion.
The protocol is fast and simple, and the results are great. What more could you want?!
The protocol is fast and simple, and the results are great. What more could you want?!
Listen to what your colleagues are saying:
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| An increasing number of scientists now prefer to use magnetic beads for their immunoprecipitation. |
Bring your research tools up to date!
Magnetic handling is easy, fast and efficient. Bound only by affinity interactions, your protein and protein complexes are preserved intact.
Other advantages of using Dynabeads® for immunoprecipitation include:
Other advantages of using Dynabeads® for immunoprecipitation include:
- No columns or centrifugations
- No time-consuming sample pre-treatment
- No loss of target protein.
- Maximum sensitivity
- Starting sample can be saliva, plasma, ascites, serum, and tissue culture or hybridoma supernatants
Shorter protocol time. Better yield and reproducibility with Dynabeads Immunoprecipitation Kit
Additional information about Immunoprecipitation
The recombinant protein A/G on the beads contains no albumin binding sites, thus avoiding co-purification of contaminating proteins.
Binding of antibodies to the beads in solution is an equilibrium reaction. To capture as many antibodies as possible, the reaction volume should be kept low to maintain high concentrations of beads and antibody.
There is no need for pre-treating the sample (even viscous samples), hence no dilution of sample or beads should be done. If the antibody concentration in the sample is suspected to be very low, the amount of beads can be increased.
As an alternative to eluting antibody from the beads, the Dynabeads® may be re-suspended in a Na-phosphate buffer (Figure 2).
To prevent co-elution, it is possible to cross-link the antibody to protein A/G on the beads prior to immunoprecipitation. This is not necessary for downstream SDS-PAGE followed by autoradiography or western blotting, and optional for Silver or Coomassie® staining.
Binding of antibodies to the beads in solution is an equilibrium reaction. To capture as many antibodies as possible, the reaction volume should be kept low to maintain high concentrations of beads and antibody.
There is no need for pre-treating the sample (even viscous samples), hence no dilution of sample or beads should be done. If the antibody concentration in the sample is suspected to be very low, the amount of beads can be increased.
As an alternative to eluting antibody from the beads, the Dynabeads® may be re-suspended in a Na-phosphate buffer (Figure 2).
To prevent co-elution, it is possible to cross-link the antibody to protein A/G on the beads prior to immunoprecipitation. This is not necessary for downstream SDS-PAGE followed by autoradiography or western blotting, and optional for Silver or Coomassie® staining.
 | |  |
| Figure 1 - Affinity purification of human IgG and immunoprecipitation of human serum albumin from 2 µl serum, using Dynabeads® Protein A. Lane 1 - Purified IgG from serum. Lane 2: Immunoprecipitated human serum albumin (cross-linking omitted thus antibody present). Lane 3: 50 x diluted serum. Lane 4: LMW. | Figure 2 - Protein elution properties |
Table 1 - Properties of the recombinant Protein A and Protein G in Dynabeads®
Dynabeads® Protein A and Dynabeads® Protein G bind with different affinities depending on the immunoglobulin type and species. Binding occurs mainly through the Fc region. The binding capacity will depend on the initial concentration of antibodies in the sample and the source of the antibody. These data are based on isolation from a sample containing 100 µg immunoglobulin per ml.
| | Protein A | Protein G |
|---|---|---|
| Source | Bacillus | E.coli |
| Molecular weight | 45 kDa | 17 kDa |
| No.of binding sites for IgG | 4 | 2 |
| Binding capacity* | 250 µg human IgG / ml beads | 640 µg mouse IgG / ml beads |
| Optimal binding pH | 7.4 | 7.4 |
True or False?
- Background can't be avoided
- Pre-clearing is necessary
- Capacity is crucial
- Slurry is cheap
Do these assumptions hold true? Myths busted here!
