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SepSphere™ Antibody/Protein/Enzyme Immobilization Kit via Glutaraldehyde

  • Image 1
  • Immobilization of protein on glutaraldehyde agarose beads
  • Schematic diagram of the workflow for protein immobilization starting with 5 mL of glutaraldehyde agarose beads
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Glutaraldehyde is reactive towards the primary amino groups of protein (including antibody and enzyme) and has been used for protein immobilization through covalent attachment to amino-activated matrixes or by mere cross-linking of protein-protein aggregates or protein adsorbed onto amino-activated matrixes (Selected review: Barbosa, O. RSC Avd. 2014, 4, 1583-1600; Fernando Lopez-gallego et al. Chapter 3 in Immobilization of Enzymes and cells: third edition, Methods in Molecular Biology, 2013, vol. 1051, p33-41). Glutaraldehyde agarose has been frequently used for protein immobilization under mild reaction conditions and is particularly suited for unstable protein.

CellMosaic designed this kit to work directly with glutaraldehyde agarose derivatized from the reaction of glutaraldehyde with amino agarose (~20 μmol amino per mL of resin). In this case, one of the aldehyde groups of glutaraldehyde is linked to the amino group of the agarose and leaves other aldehyde to react with the amino groups of the protein under very mild conditions. The final product is relatively stable due to the covalent linkage and the neighboring ionic interaction with the residual amino groups in the solid support. For example, the initial test of this kit with protein A provided a maximum loading capacity of 3 mg protein A per mL of settled beads and a binding capacity of 8 mg human IgG per mL of settled beads.

Scale of the reaction: 5 mL

Key Features of this Immobilization Kit:

  • Minimum requirement for immobilization set-up
  • All reagents, buffers, and plasticwares are included 
  • Modestly activated glutaraldehyde agarose
  • Mild reaction conditions (pH 7.0) and one step immobilization
  • Easy preparation with air push mechanism for washing: less than 2 h hands-on time

 Specification of the Solid Support

  • Functional groups: Glyoxyl
  • Matrix: 4% beaded agarose (source: GE Life Sciences, Sepharose® 4B)
  • Particle size: 45-165 um 
  • Mean bead size: 90 um   
  • Ligand concentration: 5-20 μmol glutaraldehyde per mL agarose
  • Protein loading assessed by Protein A (maximum loading): 0.07 umole per ml agarose

 Specification of the Immobilized Product

  • Immobilized chemistry: immobilized via glyoxyl agarose and reduction
  • Chemical stability: stable 
  • pH Stability: stable     

 Requirement for protein (including antibody and enzyme)

  • Preferably >90% pure by gel electrophoresis 
  • Total amount: 1-15 mg protein content as measured by UV (>15 mg is fine)

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