Immunoprecipitate using any antibody species or subclass!
Eliminate antibody band contamination of IP products.

The Thermo Scientific Pierce Direct IP Kit represents a significant advancement in immunoprecipitation (IP) technology by replacing the use of immobilized Protein A or Protein G with a method for direct covalent attachment of antibodies to the beaded agarose resin.

The primary benefits resulting from this method are the opportunity to use any species or subclass of purified antibody (not just types that bind to Protein A or G) and the ability to purify target protein without contamination by the antibody. The method also makes it possible to immunoprecipitate antigens from serum samples without co-purifying non-target immunoglobulins. Finally, the kit uses microcentrifuge spin cups to effectively wash and separate samples from the beaded agarose resin.

Highlights:

• Requires less than 10 µg of antibody – optimized procedure requires only 2-10 µg of antibody for a single IP experiment, no more than is required for traditional IP methods
• Easy and efficient scalability – use only the amount of antibody needed for a single immunoprecipitation or immobilize 100-200 µg of antibody to prepare ready-made IP affinity resin for many experiments
• Minimal antibody contamination – antibody is irreversibly attached to the agarose beads so that co-elution of heavy and light chains with the purified protein is minimized (i.e., less than 5%)
• IP with any species and subclass of antibody – Use chicken IgY, human IgE, mouse IgM or any other purified protein
• Prepared antibody affinity resin is reusable – because the antibody is covalently immobilized and not inactivated by the mild elution procedure, the resin often can be used several times
• Convenient sample handling – Pierce Spin Columns (see dimensions) eliminate resin loss and provide for efficient separation of solutions
• Complete kit – package includes cell lysis buffer and sufficient reagents and spin columns to perform at least 50 antibody immobilizations and IP experiments
• Requires purified antibody – IP antibody solution must be free of BSA, gelatin or other stabilizer proteins; for easy removal of these proteins, see our Antibody Clean-up Kit (#44600)

Applications:

• Immunoprecipitation followed by electrophoresis and excision of the protein for mass spectrometry or sequencing identification analysis
• Immunoprecipitation and SDS-PAGE analysis of a target protein whose molecular weight is similar to the heavy or light chain antibody fragment
• Immunoprecipitation of target proteins for subsequent analysis by nondenaturing methods (i.e., methods not involving SDS-PAGE)
• Immunoprecipitation with antibodies that do not bind to Protein A, Protein G or Protein A/G
• Immunoprecipitation from serum and other sample that contain immunoglobulins

Direct Immunoprecipitation Kit Details:

The Direct IP Kit method involves covalent immobilization of antibody to aldehyde-activated beaded agarose resin (AminoLink Plus Coupling Resin). By contrast with traditional IP methods, the Direct IP Kit enables elution of antigen without antibody contamination or nonspecific binding to Protein A or Protein G.

Direct immunoprecipitation in two easy steps:

Step 1: Attach antibody to agarose resin
The first step in the Direct IP Method is to prepare the IP antibody affinity resin. Pure antibody (see Highlights) is incubated in phosphate buffer with an appropriate volume of AminoLink Plus Coupling Resin, an aldehyde-activated beaded agarose resin. During the incubation, various lysine epsilon-amines on the surface of the large antibody molecule react with aldehyde groups on the resin to form Schiff-base bonds that are then stabilized to secondary amine bonds in the presence of cyanoborohydride (see details). The immobilization reaction, known as reductive amination, is nondenaturing and typically results in greater than 85% antibody coupling efficiency with minimal loss of antigen-binding function.

Step 2: Perform immunoprecipitation experiments
Once prepared, the antibody resin can be used in several parallel aliquots or in total for IP experiments. Antibody resin is incubated with a sample that contains the protein antigen of interest, allowing the antibody:antigen complex to form. After washing to remove nonbound (presumably undesired) components of the sample, the antigen is recovered by dissociation from the antibody with elution buffer supplied in the kit. The entire procedure is performed in a microcentrifuge spin cup, allowing solutions to be fully separated from the agarose resin upon brief centrifugation. Only antigen is eluted by the procedure, enabling it to be identified and further analyzed without interference from antibody fragments. Furthermore, the antibody resin can be reused for additional rounds of immunoprecipitation.

How exactly is the Direct IP Method different from traditional IP?
See a side-by-side comparison of traditional and Direct IP Kit immunoprecipitation methods.

Will the Direct IP Method always work better than a traditional IP method?
There are certain atypical circumstances in which the Direct IP Method will not work as well as a traditional IP method. These include one of the following two situations, which are peculiar to the specific antibody being used in the procedure:

• The covalent immobilization completely inactivates the antibody. As a result, little or no antigen will be immunoprecipitated despite committing ample antibody to the procedure.
• The antibody:antigen affinity interaction is so strong that the kit Elution Buffer does not efficiently dissociate the target protein antigen from the covalently immobilized antibody. As a result, the antigen can be recovered only by using harsher elution conditions, which may have the undesirable effect of denaturing and eluting fragments of the antibody.

Which of the three different kinds of Pierce IP Kits is best for me?
Compare the features and requirements of Thermo Scientific Pierce Classic, Crosslink and Direct IP Kits to determine which one is most appropriate for an intended IP experiment and available source of antibody.

Related Products:

Classic IP Kit – for traditional IP using Protein A/G Agarose
Crosslink IP Kit – for antibody immobilization by crosslinking
Co-IP Kit – for co-immunoprecipitation using the Direct IP method

Antibody Clean-up Kit – for removing BSA or gelatin from antibody samples before immobilization
Control Agarose Resin – for pre-clearing samples and various negative controls for IP
Spin Columns - Screw Cap – the same spin columns (#69705) supplied in the kit
Pierce IP Lysis Buffer – best cell lysis buffer for immunoprecipitation
M-PER Mammalian Protein Extraction Reagent – alternative, IP-compatible cell lysis buffer
AminoLink Plus Immobilization Kit – for large scale antibody immobilization

References:

1. Kwon, Y.H., et al. (2002) J. Biol. Chem. 277, 41417-41422.
2. Eberhardt, W., et al. (2002) Mol. Endocrinol. 16, 1752-1766.
3. Pritzker, L.B., et al. (2004) Mol. Biol. Cell. 15, 2834-2841.
4. Su, Y., et al. (2003) J. Biol. Chem. 278, 21013-21018.