Cyanogen bromide (CNBr) activation of agarose beads is a time-tested conventional method of immobilizing protein and antibody on resin for affinity purification. CNBr reacts with hydroxyl groups on agarose to form cyanate esters or imidocarbonates. These groups react with primary amines of proteins under mild conditions, resulting in covalent immobilization of the protein. The preferred linkage is imidocarbonate, which has multiple point attachments and no net charge. Some products may have an isourea bond product, which is somewhat unstable and may result in leakage of the coupled ligand and cause non-specific binding. CNBr is highly hazardous and requires highly trained personnel to use it. The activation and immobilization also require many specialized buffers.

CellMosaic designed this kit to work directly with CNBr-activated agarose so that hazardous CNBr is not involved in the reaction. The CNBr-activated agarose is stabilized with other additives, and is stable at 2-8°C for years without losing its activity. The protocol is designed to maximize the amount of stable imidocarbonate product produced and minimize the unstable isourea product. 

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 
• Stabilized cyanogen bromide-activated agarose for high loading
• Easy preparation with air push mechanism for washing: less than 2 h hands-on time

 Specification of the Solid Support

• Functional groups: CNBr-activated (cyanate esters or imidocarbonates)
• Matrix: 4% beaded agarose (source: GE Life Sciences, Sepharose® 4B)
• Particle size: 45-165 um 
• Mean bead size: 90 um   
• Ligand concentration: N/A
• Protein loading assessed by Protein A (maximum loading): 0.14 umole per ml agarose

 Specification of the Immobilized Product

• Immobilized chemistry: immobilized via CNBr activated agarose
• Chemical stability: all commonly used aqueous buffers
• pH Stability: long term: 3 to 9; short term: 2 to 10
• Physical stability: negligible volume variation due to changes in pH or ionic strength    

 Requirement for protein (including antibody and enzyme)

• Preferably >90% pure by gel electrophoresis 
• Total amount: 1.3-30 mg protein content as measured by UV and depend on your loading requirement (>30 mg is fine)