The Thermo Scientific Pierce Crosslink IP Kit adapts the traditional IP method to include reagents and protocol for crosslinking IP antibodies to Protein A/G agarose to enable antigen immunoprecipitation without antibody contamination.
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.
Features of the Crosslink IP Kit:
• 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)
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.
Sufficient For: Preparing and performing at least 50 IP reactions, each with 10 µL of resin • Protein A/G Plus Agarose, 0.55 mL • DSS Crosslinker, 8 x 2 mg • Coupling Buffer (20X), 25 mL • IP Lysis/Wash Buffer, 2 x 50 mL • Conditioning Buffer (100X), 5 mL • Tris-Buffered Saline (20X), 25 mL • Elution Buffer, 50 mL • Lane Marker Sample Buffer (5X), 5 mL • Control Agarose Resin, 2 mL • Spin Columns and Collection Tubes