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 immunoprecipitation experiment and available source of antibody.

What is immunoprecipitation?

Immunoprecipitation (IP) is small scale affinity purification of antigen using a specific antibody. Traditional IP involves incubation of antibody with a protein mixture containing the antigen, followed by capture of the antibody:antigen complex to Protein A or Protein G immobilized on beaded agarose resin. After washing to remove nonbound (presumably undesired) components of the sample, antibody and antigen are recovered by boiling the beaded affinity resin in sample loading buffer for analysis by SDS-PAGE.

How are the Thermo Scientific Pierce IP Kits different from traditional IP?

The three main kinds of Pierce IP Kits, like the traditional IP method, require a specific antibody and use beaded agarose affinity resin for immunoprecipitation of the antigen. Unlike in the traditional IP method, all three Pierce IP Kits provide a complete set of reagents and convenient spin columns and collection tubes to perform successful IP experiments with ease. The Crosslink and Direct IP Kits comprise two different methods for covalent immobilization of the antibody to the agarose resin, enabling purification of antigens without the usual co-elution of the antibody heavy and light chain fragments.

What is the key difference between the Crosslink and Direct IP Kits?

Both the Crosslink IP Kit and the Direct IP Kit allow covalent attachment of the IP antibody to the agarose resin, enabling purification of antigen without the usual co-elution of antibody heavy and light chain fragments.

• The Crosslink IP Method uses crosslinking to covalently attach the antibody to the Protein A/G Agarose Resin. As such, the method requires an IP antibody of a species and subclass that can be bound by either Protein A or Protein G. (See Protein A and Protein G binding characteristics by antibody species and class).
• The Direct IP Method uses reductive amination to directly couple antibody primary amino groups to an aldehyde-activated agarose support called AminoLink Plus Coupling Resin. Because the coupling reaction occurs to any primary amine, the antibody must be pure – that is, it must be free of amine-containing buffers (e.g., Tris) and all other proteins such as BSA and gelatin. Although pure antibody is required for the Direct IP Method, there are no species or subclass requirements because coupling is not dependent on Protein A or Protein G binding affinities. Chicken IgY, human IgE and IgM, or any purified protein can be immobilized to the AminoLink Plus Coupling Resin.

How do these different IP methods affect results?

Comparison of Thermo Scientific Pierce IP Kit methods. Identical amounts of affinity-purified goat anti-GFP antibody and and cell lysate were used with identical amounts of respective agarose beads for purification by the three different kit methods. E1-E3 denote three successive elution fractions electrophoresed on a 12% polyacrylamide reducing gel and stained with GelCode Blue Stain Reagent. MW = Molecular Weight Marker.

Related Thermo Scientific Pierce Products: