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Pierce Crosslink IP Kit


Traditional IP combined with crosslinking technology and spin column sample-handling.

Thermo Scientific Crosslink IP KitThe 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.

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)

Product Details:

Crosslink IP Kit uses DSS to attach antibody to agarose beads

Antibody immobilization method of the Pierce Crosslink IP Kit. The 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 (Part No. 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. Crosslink IP Kit immunoprecipitates without antibody contamination

References:

  1. Rubenstein R. C. et al. (2011) Regulation of endogenous ENaC functional expression by CFTR and {delta}F508-CFTR in airway epithelial cells. Am J Physiol Lung Cell Mol Physiol. 300, L88-101.
  2. Qian W. et al. (2011) Regulation of the alternative splicing of tau exon 10 by SC35 and Dyrk1A. Nucleic Acids Res., gkr195.
  3. Kannan Y. et al. (2011) I{kappa}B{zeta} augments IL-12- and IL-18-mediated IFN-{gamma} production in human NK cells. Blood. 117, 2855-63.
  4. Armstrong J. L. et al. (2011) Oncogenic B-RAF signaling in melanoma impairs the therapeutic advantage of autophagy inhibition. Clin. Cancer Res. 17, 2216-26.
  5. Liu T. et al. (2010) Regulation of vimentin intermediate filaments in endothelial cells by hypoxia. Am J Physiol Cell Physiol. 299, C363-73.

Related Resources:

Review of immunoprecipitation techniques and methods
Pierce IP Kit Selection Guide

Related Products:

Other immunoprecipitation kits – agarose, magnetic, co-IP, etc.

Control Agarose Resin – for pre-clearing samples and various negative controls for IP
Pierce IP Lysis Buffer – best cell lysis buffer for immunoprecipitation
Protein A/G Agarose and UltraLink Resins
DSS Crosslinker


Ordering Information
 
Product # Description Pkg. Size Instructions MSDS CofA Price
26147 Pierce Crosslink IP Kit
Formulation: Multi-component kit
Sufficient For: Preparing and performing at least 50 IP reactions, each with 10µL of resin

Kit Contents:
Protein A/G Plus Agarose, 0.55mL
DSS Crosslinker, 8 x 2mg
Coupling Buffer (20X), 25mL
IP Lysis/Wash Buffer, 2 x 50mL
Conditioning Buffer (100X), 5mL
Tris-Buffered Saline (20X), 25mL
Elution Buffer, 50mL
Lane Marker Sample Buffer (5X), 5mL
Control Agarose Resin, 2mL
Spin Columns and Collection Tubes
50-rxn kit Product Instructions for product #26147 Pierce Crosslink IP Kit MSDS for product #26147 Pierce Crosslink IP Kit Certificate of Analysis for product #26147 Pierce Crosslink IP Kit $323.00

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Instructions | MSDS | CofA
Product Instructions | MSDS | CofA  

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