Update profile
    Change password
    Sign out

GlycoLink IP Kit


Immunoprecipitate using antibodies immobilized by their Fc-region glycosylation.

GlycoLink Immunoprecipitation Kit

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:

General structure and reaction scheme for the Thermo Scientific GlycoLink Immobilization for immobilization of protein at carbohydrates.
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 STAT1 and EGFR using polyclonal antibody immobilized at Fc region
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).

More Product Data

General References:

  1. Abraham, R., et al. (1991). The influence of periodate oxidation on monoclonal antibody avidity and immunoreactivity. J. Immunol. Methods 144(1):77-86.
  2. Byeon, J.Y., et al. (2010). Efficient bioconjugation of protein capture agents to biosensor surfaces using aniline-catalyzed hydrazone ligation. Langmuir 26(19):15430-5.
  3. Dirksen, A., et al. (2006). Nucleophilic catalysis of hydrazone formation and transimination: implications for dynamic covalent chemistry. J. Am. Chem. Soc. 128(49):15602-3.
  4. Dirksen, A., Dawson, PE. (2008). Rapid oxime and hydrazone ligations with aromatic aldehydes for biomolecular labeling. Bioconjug. Chem. 19(12):2543-8.
  5. Domen, P., et al. (1990). Site-directed immobilization of proteins. J. Chromatogr. 510:293-302.
  6. O’Shannessy, D., et al. (1984). A novel procedure for labeling immunoglobulins by conjugation to oligosaccharide moieties. Immunol. Lett. 8:273-7.
  7. Zing, Y., et al. (2009). High-efficiency labeling of sialylated glycoproteins on living cells. Nat. Methods 6(3):207-9.

Related Resources:

GlycoLink FAQ
Review of glycosylation and methods to analyze glycoproteins and glycopeptides
Review of affinity purification
Review of covalent immobilization methods
Hydrazide crosslinker chemistry

Related Products:

GlycoLink Immobilization Kits and Resins
GlycoLink Coupling Catalyst
Sodium meta-Periodate
Hydrazide- and Alkoxyamine-activated Biotinylation Reagents
Glycoprotein Carbohydrate Estimation Kit


Ordering Information
 
Product # Description Pkg. Size Instructions MSDS CofA Price
88943 GlycoLink IP Kit
Formulation: Multi-component kit with UltraLink Hydrazide Resin
Sufficient For: 25 IP/Co-IP reactions, each using 20µL antibody-coupled resin


Kit Contents:
UltraLink Hydrazide Resin, 0.5mL
GlycoLink Coupling Buffer, 50mL
Aniline, 0.2mL
Sodium meta-periodate, 0.5g
Wash Buffer, 60mL
Zeba Spin Desalting Columns,0.5mL, 25 columns
IP Lysis/Wash Buffer, 50mL
PBS Pack (makes 500mL), 1 pack
Elution Buffer, pH 2.8, 50mL
Lane Marker Sample Buffer, Non-reducing, (5X), 5mL
Pierce Spin Columns-Screw Caps, 25 columns
Collection Tubes, 2mL, 100 tubes
25-rxn kit Product Instructions for product #88943 GlycoLink IP Kit MSDS for product #88943 GlycoLink IP Kit Certificate of Analysis for product #88943 GlycoLink IP Kit $433.00

Add to cart

Instructions | MSDS | CofA
Product Instructions | MSDS | CofA  

Recommend this page

Follow Us
Email Sign-up  Email Announcements

You may also need
88223 - HisPur Ni-NTA Resin

 



Antibodies  |   Molecular Biology   |   Cell Biology   |  Thermo Scientific  |   * Trademarks  |   Privacy Statement
© 2014 Thermo Fisher Scientific Inc.   |   3747 N Meridian Rd, Rockford, IL USA 61101   |   1-800-874-3723  or  815-968-0747