Dynabeads™ Protein A are uniform, 2.8 µm superparamagnetic beads with recombinant Protein A (~45 kDa) covalently coupled to the surface. Dynabeads Protein A provide a superior alternative to Sepharose™ resin or agarose resin for immunoprecipitation (IP), and both manual and automated protocols are available.
• IP in less than 40 minutes
• High target protein yield with low antibody consumption
• Very low non-specific binding with high signal-to-noise ratio
• No columns, centrifugations, or time-consuming pre-clearing required
• High reproducibility and high throughput compatible with KingFisher™ instruments
Manual Dynabeads separation is fast and easy to perform
The manual protocol is simple and can be performed in under 40 minutes. First, the antibody for the target protein is incubated with the Dynabeads Protein A in a tube for 10 minutes. Excess antibody is washed away by placing the tube in a DynaMag™ magnet and removing the supernatant. The antibody-coated beads can then be used for a variety of downstream applications including IP, Co-IP, chromatin IP (ChIP), RNA IP (RIP), small-scale IgG purification, and protein purification. Bound material is easily collected using a DynaMag magnet due to the unique magnetic properties of the Dynabeads. The recombinant protein A on the beads contains no albumin binding sites, thus albumin is not co-purified during the procedure. The IP is fast and gives high yield, high reproducibility, and very little non-specific binding, thus pre-clearing is not required.
Automated Dynabeads separation helps increase throughput and reduces hands-on time
If you are working with several samples in parallel, the number of washing steps and the hands-on time increases proportionally with the number of samples. Pipetting and other manual handling tend to be less consistent than automation when working with many samples at a time. To better handle a medium- to high-throughput number of samples, reduce hands-on time, and secure high reproducibility, we have developed IP protocols for the KingFisher Flex and KingFisher Duo Prime instruments. The automated protocols replicate the manual protocols, obtaining equally high target protein yield and the same low non-specific binding and high reproducibility. It doesn’t matter if you are working with 10 or 96 samples, the IP protocol is less than 40 minutes regardless. Just load the reagents on the plates, push the “Start” button and by the time you have prepared for downstream analysis, the IP is done. Some optimization (e.g., incubation times) might be necessary depending on your antibody and the abundance and/or specificity of your target protein.
Gentle separation causes minimal physical stress to proteins
The magnetic separation technology utilized by Dynabeads Protein A is rapid and gentle, causing minimal physical stress to your target proteins. This permits the isolation and concentration of labile composites that might otherwise dissociate or be damaged by proteases during long incubation times. Native protein conformation and large protein complexes are preserved.
Binding strength and capacity
Dynabeads Protein A allow for isolation of most mammalian immunoglobulins (Ig). The amount of Ig captured depends on the concentration of Ig in the starting sample and on the type and source of the Ig. 100 µL of Dynabeads Protein A will isolate approximately 25–30 µg human IgG from a sample containing 20–200 µg IgG/mL. Predominant Fc-binding allows optimal Ig orientation. The antibodies bind to the outer smooth surface of the beads, thus are not trapped in large pores as with Sepharose/agarose-based beads. All antibodies are available for protein binding, so low amounts of antibody are required while still obtaining the same high yield of target protein. The smooth bead surface is also responsible for the low non-specific binding that Dynabeads are known for.
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