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Immunoprecipitation Kit Selection Guide

Compare the features and requirements of Classic-, Crosslink- and Direct-style Thermo Scientific Pierce IP and Co-IP Kits to determine which one is most appropriate for an intended immunoprecipitation experiment and available source of antibody. All three types of kits are available in agarose and magnetic bead formats.

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Traditional IP methodology

Immunoprecipitation (IP) is the small-scale affinity purification of antigen using a specific antibody. Traditional IP strategies involve incubating an antibody with a protein mixture containing the antigen, followed by capturing the antibody:antigen complex with Protein A or G immobilized to beaded agarose resin. After washing the resin to remove nonbound and 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.

The traditional procedure is performed in a microcentrifuge tube, requiring that the solution be carefully withdrawn from the agarose resin after being pelleted by centrifugation. Both the target antigen and the antibody are eluted in the final fraction, resulting in the presence of prominent heavy and light chain antibody bands in the electrophoresis gel. If the target protein has a molecular weight that is similar to one of the contaminating antibody fragments, its presence may be masked, making it impossible to identify or further characterize the immunoprecipitated product.

The same method can be done using magnetic particles instead of agarose resin. Instead of centrifuging to separate beads and solutions at various steps, a magnet is used to gather and "pellet" the small particles from suspension so that solutions can be decanted or pipetted.

Immunoprecipitation (IP) Techniques and Methodology

Tech Tip #64: Immunoprecipitation technical guide and protocols

Overview of Solid Supports for Affinity Purification

 

 

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Pierce Magnetic Beads

How Pierce IP Kits differ from traditional IP

The three kinds of Thermo Scientific 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, though, 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. Aspects of each kit that differ from traditional IP protocols are as follows:

With the Classic IP Kit method, the antibody is captured by Protein A/G Agarose resin similar to the traditional IP method. The antibody and antigen, though, are efficiently dissociated from the Protein A/G Agarose resin with elution buffer (pH 2.8) instead of being denatured or cleaved into separate subunits with the traditional IP method, affording greater flexibility in subsequent analytical strategies. Furthermore, the affinity resin often can be reused for additional rounds of immunoprecipitation.

The Crosslink IP Kit and Direct IP Kit employ 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.

  • The Crosslink IP Kit 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. Compare Protein A and Protein G binding characteristics.
  • The Direct IP Kit 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 at 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 Kit, 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. (Our Direct Magnetic IP Kits use NHS-activated Magnetic Beads to accomplish the same sort of amine-based immobilization).

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Classic IP Kit
Crosslink IP Kit
Direct IP Kit


Classic Magnetic IP Kit
Crosslink Magnetic IP Kit
Direct Magnetic IP Kit


All IP and Pull-Down Kits


Protein A/G Agarose Resins
Protein A/G Magnetic Beads

AminoLink Plus Coupling Resin
NHS-Activated Magnetic Beads

Also try the
GlycoLink IP Kit
(Part No. 88943)

This kit provides an alternative "direct-style" method ideal for polyclonal antibodies. The IP antibody is immobilized by its sugar groups (glycosylation). Therefore, its antigen-binding activity is more fully retained.

Comparison of IP methodologies. Click image to enlarge.

Comparison of immunoprecipitation kit methods

How different immunoprecipitation methods affect IP yield and purity

Comparison of results using Thermo Scientific Pierce Direct, Crosslink and Classic Kits. Comparison of results using Thermo Scientific Pierce IP Kits. 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.

 

Comparision of IP method features. The following table compares the key features of traditional "do-it-yourself" immunoprecipitation techniques to the Thermo Scientific Pierce IP Kits. Consideration of these features can help to determine which method is most appropriate for the available reagents and downstream application. Also available is the Thermo Scientific GlycoLink IP Kit, an alternative direct-IP method for polyclonal antibodies (i.e., glycoproteins).
Feature Traditional
IP Method
Pierce
Direct IP Kits
Pierce
Crosslink IP Kits
Pierce
Classic IP Kits
Agarose bead kits
(and stand-alone resins)
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Agarose Resins
Part No. 26148
(AminoLink Plus)
Part No. 26147
(Protein A/G Plus)
Part No. 26146
(Protein A/G Plus)
Magnetic bead kits
(and stand-alone particles)
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Magnetic Beads
Part No. 88828
(NHS Activated)
Part No. 88805
(Protein A/G Beads)
Part No. 88804
(Protein A/G Beads)
Crosslinker-mediated immobilization No No Yes
(DSS)
No
Requires purified antibody in amine-
free and protein-free storage solution
No Yes No No
Antibody is covalently attached to agarose resin No Yes Yes No
Antibody is oriented
Yes No Yes Yes
Antibody elutes with antigen
Yes No No Yes
Antigen recovery method
Boiling w/ SDS
(Low pH)
Low pH elution Low pH elution Low pH elution
(Boiling w/ SDS)
Relative antigen recovery ²
Variable High Medium Highest
Immobilized antibody can be reused No Possible ³ Possible ³ No
1 Commercially available resins vary in binding capacity and performance for IP assays.
2 Antigen yield is depends on the activity of the antibody, specific binding conditions and the immobiliztion method used.
3 It is possible to reuse the prepared antibody affinity resin if the antibody remains functional following low pH elution.
Browse all Thermo Scientific Pierce Immunoprecipitation Kits

Instructions | MSDS | CofA
Product Instructions | MSDS | CofA  

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