Recommended IP method for protein sample sizes <2 mL
While agarose/Sepharose® slurry and columns perform well for purifications of large amounts of protein or antibody, magnetic beads are best suited for the more-common, smaller-scale isolation of specific proteins and protein complexes. Protein research needs have changed since the introduction of Sepharose® in the 1970s, with the keys being a balance of capacity/yield, reproducibility, purity, and cost.
There are many myths about IP that we easily break down for you here in these IP Myth videos. If you are looking for an alternative guide to help you find the the optimum IP product, try this interactive video selection guide.
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Find the right immunoprecipitation product for your experiment
Choose this if you've got an antibody that recognizes your protein
Your choice of antibody binding products depends on whether your downstream assay is mass spectrometry, or if you don’t want the antibody co-eluted with your target protein, etc.
Antibody binding is the most common method and is used when your target antibody can be bound directly to the beads or indirectly to a pre-coated ligand on the magnetic beads.
Antibody binding product selection guide
||Protein A, G, or L
|Surface- activated beads (epoxy)*
||Non-covalent antibody binding
||Non-covalent antibody binding
||Covalent antibody binding
|Antibody co-eluted off the beads
|Type of ligand
||Different ligands bind different species and antibody subclasses with different specificity‡
||Anti-mouse binds mouse IgG1, IgG2a, IgG2b; anti-rabbit binds all rabbit IgGs
|Mass spec compatible
|Coupling time / temp
- Protein A: 10 min, room temp
- Protein G: 10–40 min, room temp
- Protein A/G: 60 min, room temp
- Protein L: 60 min, room temp
|>30 min / 2–8°C
||16–24 hr, 37°C
*See more choices in surface-activated Dynabeads® for the binding and capture of additional targets.
†Crosslinking can be performed to avoid co-elution of the antibody, but this can decrease the yield of the target antigen.
‡Check the compatibility of your antibody with our kits using the Antibody Compatibility Table.
Choose this if you've got a biotinylated antibody (or ligand) that recognizes your protein
Main advantages for using a biotinylated antibody with streptavidin-coated beads for IP
- If you have a sample rich in soluble IgGs
- If you have a recombinant antibody lacking Fc regions
- If you already have streptavidin-coated Dynabeads® in the lab
- If you need a bead compatible with mass spectrometry (secondary-coated and epoxy-coated Dynabeads® are also compatible with mass spectrometry)
To allow flexibility, four different types of streptavidin-coupled Dynabeads® are available. View the Dynabeads® streptavidin selection guide to see the different characteristics.
Over the past 25 years, streptavidin-coupled Dynabeads® in combination with a biotinylated ligand have been widely used and are cited in thousands of papers for diverse manual and automated applications. The most widely used application is for purification of nucleic acids (NA), but it is also used for immunoprecipitation (IP).
Choose this if you've got a recombinant (fusion tagged) protein
Popular fusion tags for recombinant protein expression
- His tag—consists of a string of six to nine histidine residues; primarily used for purification via immobilized metal affinity chromatography (IMAC)
- GST tag—consists of glutathione S-transferase (GST), a complete 211 amino acid protein (26 kDa); primarily used for purification via glutathione agarose resin
- HA tag—consists of a peptide (YPYDVPDYA) derived from the human influenza hemagglutinin (HA) protein; immobilized anti-HA antibodies are used to immunoprecipitate HA-tagged recombinant proteins
- c-Myc tag—consists of a peptide (EQKLISEEDL) derived from the human c-myc oncogene (p62 c-myc); immobilized anti-c-Myc antibodies are used to immunoprecipitate c-Myc-tagged recombinant proteins.
His and GST are not true epitope tags, because they are not usually purified or detected via specific antibodies. By contrast, HA and c-Myc tags are true epitope tags, because their only means of purification or detection is via specific antibodies. Epitope tags are seldom used for bulk purification but are most often used for immunoprecipitation or co-immunoprecipitation (IP, co-IP). Nevertheless, all four of these tag systems can be used for either purification or pull-down applications.
If you’ve got a biotinylated antibody that recognizes the protein you wish to capture, you can create your own affinity product with one of these products.
Dynabeads® magnetic beads comparison data
In comparison studies with Dynabeads® magnetic beads and immunoprecipitation kits from other manufacturers, kits offer excellent purification characteristics and experimental reproducibility in a shorter protocol time.
Shorter protocol time, better yield and reproducibility with Dynabeads® Immunoprecipitation Kit
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: Low-molecular weight markers.
Dynabeads® Protein A Immunoprecipitation Kit vs. alternative suppliers
Immunoprecipitation was performed on Daudi cell lysate using 5 μg antibody with all products. 10% eluate from each IP experiment was loaded on a NuPAGE® 4–12% Bis-Tris and electrophoresed using MES running buffer. The resulting gel was stained using the SilverQuest ™ Silver Staining Kit (see below).
Protein elution properties
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. 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.
The publication trend for immunoprecipitation using magnetic separation is skyrocketing
- Higher reproducibility—handling consistency leads to more consistent results
- Easier handling—~30 min protocol and no centrifugation helps make magnetic beads popular for those doing IP on a regular basis
- Almost no background—magnetic beads have minimal non-specific binding, and no pre-clearing is necessary
- Antibody savings—all binding occurs on bead outer surface, helping you conserve precious antibodies
- Lower signal-to-noise—easy and efficient washing helps ensure superior contaminant removal
- Gives you more control—designed to help you balance capacity/yield, reproducibility, and purity and reduce overall cost
Browse our list of Dynabeads® magnetic beads citations
Dynabeads® IP citations: all sources
Dynabeads® IP citations: Nature
Dynabeads® ChIP citations: all sources
Dynabeads® ChIP citations: Nature
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*Note for mobile users: the interactivity of the Selection Guide for Immunoprecipitation does not function on mobile devices. To step through that video-based selection guide in your mobile device, use the links in the video description area (found below video play window).