Purify target protein complexes without antibody interference!

The Thermo Scientific Crosslink IP Kit extends the functionality of traditional immunoprecipitation (IP) methods by adding crosslinking technology and microcentrifuge spin column sample handling to the procedure. The primary benefits resulting from these features are the ability to purify target protein without contamination by the antibody and the ability to more effectively wash and separate samples from the beaded agarose resin.

Highlights:

• Eliminates 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 
• Easy and efficient scalability – use only the amount of antibody needed for a single IP experiment or immobilize 100-200 µg of antibody to prepare ready-made IP affinity resin for many experiments
• Assay reliability and sample handling – Pierce Spin Columns eliminate resin loss and provide for more efficient separation of solutions than traditional IP methods that use only microcentrifuge tubes 
• Prepared antibody affinity resin is reusable – because the antibody is covalently immobilized and usually is not inactivated by the mild elution procedure, the resin often can be used several times 
• Complete kits – package includes sufficient reagents and spin columns to perform at least 50 antibody immobilizations and IP experiments

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 Kit Details:

The Thermo Scientific Pierce Crosslink IP Kit uses DSS to covalently attach antibody to Protein A/G beaded agarose resin. By contrast with traditional IP methods, the Classic IP Kit provides for antigen elution without antibody contamination.

The Pierce Crosslink IP Kit method involves capturing the IP antibody to Protein A/G Agarose resin and covalently immobilizing it to the support by crosslinking with disuccinmidyl suberate (DSS). The antibody resin is then incubated with the 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 often can be reused for additional rounds of immunoprecipitation.

Purify antigen without antibody intereference using the Thermo Scientific Pierce Crosslink IP Kit. E. coli cells expressing fusion of green fluorescent protein (GFP) were extracted with B-PER Bacterial Protein Extraction Reagent (Product # 78248) and then immunoprecipitated using a polyclonal goat anti-GFP antibody. Eluted IP products were separated by SDS-PAGE and coomassie stained (GelCode Blue Stain Reagent) to detect total protein. Lane 1 shows the single product obtained using the Crosslink IP Kit. Lane 2 displays the multiple antigen and antibody fragments obtained using a traditional IP method. MW is a molecular weight marker.

How can I be sure that the Protein A/G Agarose will bind to my antibody?
Protein A/G is a recombinant protein that combines antibody-binding domains from Protein A and Protein G. Typically, Protein A is preferred for use with Rabbit polyclonal antibodies, while Protein G is preferred for use with mouse antibodies (especially monoclonals of the IgG1 subclass).
Compare Protein A, Protein G and other antibody-binding proteins
Look up Protein A/G binding characteristics by antibody species and class

Will the Crosslink IP Method always work better than a traditional IP method?
There are certain atypical circumstances in which the Crosslink 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 antibody is very unstable and is inactivated by the covalent crosslinking step. As a result, little or no antigen will be immunoprecipitated despite committing ample antibody to the procedure. In this case, use a corresponding Classic IP Kit if covalent attachment of the antibody is not absolutely necessary; use the Direct IP Kit if an alternative method of covalent attachment is needed. 
• 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. In this case, no method involving covalent attachment of the antibody will be effective, and the best option is to use the Classic IP Kit.

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.

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References:

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