Immunoprecipitate using antibodies immobilized by their Fc-region glycosylation.
The Thermo Scientific GlycoLink IP Kit provides components for effective immunoprecipitation (IP and co-IP) based on small-scale, covalent, affinity-resin immobilization of antibodies and other glycoproteins via oxidized sugar groups.
The GlycoLink Kit uses hydrazide-activated UltraLink Resin to immobilize and perform 25 IP or Co-IP assays with different antibodies or glycoprotein ligands. The method is ideal for polyclonal antibodies, which typically have abundant carbohydrates (glycosylation) on their Fc portions. Monoclonal antibodies that contain adequate carbohydrates are also effective. Antibodies immobilized by this hydrazide-to-carbohydrate method have unobstructed antigen-binding sites and optimal purification capability. The prepared glycoprotein or antibody columns can be regenerated and reused at least five times for immunoprecipitation, co-immunoprecipitation or pull-down affinity-purification without significant loss in binding capacity.
Highlights:
Efficient immobilization – couple 2 to 10µg of oxidized antibody or other glycoprotein to 20µL of resin in 2 hours or less
Stable immobilization – resonance structure of the hydrazone bonds are sufficiently stable so the antibody remains coupled during elution
Preserves binding function – immobilizes IgG via the Fc region, thereby keeping both antigen binding sites available for capturing target
Versatile applications – immobilize any molecule that contains oxidizable sugars, including glycoproteins for use in purifying protein interaction binding partners
Applications:
Immunoprecipitation followed by electrophoresis and excision of the protein for mass spectrometry or sequencing
Immunoprecipitation and SDS-PAGE analysis of a protein with a molecular weight similar to the antibody’s heavy or light chain
Identification of binding partners of immobilized glycoprotein
Co-immunoprecipitation for determining protein-protein interactions
Includes:
Kit contains resin, buffers and reagents for immobilization; desalting columns; IP lysis/wash and elution buffers for immunoprecipitation; microcentrifuge spin columns and collection tubes; and loading buffer for SDS-PAGE
Product Details:
Chemistry of antibody immobilization with Thermo Scientific GlycoLink Resin. Polyclonal antibodies with oxidized sugars contain aldehyde groups which will conjugate to the hydrazide-activated beaded resin support. Learn more about carbonyl-reactive immobilization methods and about hydrazide crosslinking chemistry.
The GlycoLink IP Kit method results in covalent attachment of oxidized sugar groups of an IP antibody (or any other glycoprotein with oxidizable carbohydrate groups) to the hydrazide resin. 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 non-bound 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 resin with a brief centrifugation. Only the antigen is eluted by the procedure, enabling it to be identified and further analyzed without interference from antibody fragments.
Immunoprecipitation of large targets using carbohydrate-immobilized IgG. Purified rabbit polyclonal anti-STAT1 or rabbit polyclonal anti-EGFR (Part No. PA1-1110) was oxidized and coupled to 20µL of resin. A549 cell lysate (0.5mg) was incubated with the coupled resin for 1 hour at room temperature. Eluted proteins were resolved on a 4-20% SDS-PAGE Gel, transferred to a nitrocellulose membrane and probed with the appropriate antibodies. Detection was performed using SuperSignal West Dura Chemiluminescent Substrate (Part No. 34075) and a CCD Imager (10 second exposure).
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