Immunoprecipitation using Dynabeads® Protein A or G
Immunoprecipitation is going magnetic!
Agarose/Sepharose® slurry performs well in the purification of large amounts of protein or antibody, but it is not well suited for the smaller-scale isolation of specific proteins and protein complexes.
For this, Dynabeads® magnetic beads offer the quality and specificity you need, and many scientists across the globe are already taking advantage of the benefits of magnetic bead–based isolation—Dynabeads® are used and cited in over 5,200 published articles.
Are you still using Sepharose®/agarose slurry?
Thousands of scientists are moving away from Sepharose® and agarose and are adopting magnetic beads.
Figure 1. Dynabeads® magnetic separation is the fastest growing method for immunoprecipitation (IP) and chromatin immunoprecipitation (ChIP). Graphs show percent increase/decrease of published papers using agarose (green), sepharose (blue) and Dynabeads® (red) over the last 7 years (IP) and 5 years (ChIP). Source: Google Scholar, Sept. 2013. Sepharose® is a trademark of GE Healthcare companies.
What is most important for immunoprecipitation?
With a fast and simple protocol and superior results, it's easy to see the benefits of Dynabeads® for immunoprecipitaion.
Minimal nonspecific binding
Easy and efficient washing
Magnetic separation protocol
Binding occurs on outer surface
|Time and effort
30 minutes start to finish
Product and handling consistency
Read what your colleagues have been saying
An increasing number of scientists now prefer to use magnetic beads for their immunoprecipitation.
60% of scientists asked indicate they will start using magnetic technology within 1–3 years.
(Source: Survey conducted in December 2010, sample size of 1,013 scientists).
Avoid these two typical problems:
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:
- 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
Figure 2. Dynabeads® magnetic beads can help you achieve highly pure isolates in a short protocol time.
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 milliliter.
|Protein A||Protein G|
|Molecular weight||45 kDa||17 kDa|
|No.of binding sites for IgG||4||2|
|Binding capacity*||250 µg human
|640 µg mouse
|Optimal binding pH||7.4||7.4|
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 3). 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 3. Protein elution properties.
Affinity purification of human IgG
Figure 4. 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: 50X diluted serum.
Lane 4: LMW.
IP protocol comparison