PureCube Amine Activated Agarose is available as off-the-shelf item and as a bulk product. Our careful in-house production provides low lot-to-lot variation, making activated agaroses also suitable for commercial production. PureCube Amine Activated Agarose carries an amine functionality. Proteins can bind covalently via free carboxy groups using the EDC/NHS activation chemistry.
PureCube Amine Activated Agaroses provide:
||Coupling proteins via direct covalentvia EDC
||Free Carboxy Groups
||Delivered as a 50 % suspension
||Affinity agarose with activated Amine groups
|Required Buffers & chemicals
- Coupling buffer I (PBS): 150 mM Na phosphate, 100 mM NaCl, pH 7.2
- Coupling buffer II: 0.1 M MES, 150 mM NaCl, pH 4.7
- Wash buffer I (PBS): 150 mM Na phosphate, 100 mM NaCl, pH 7.2
- Wash buffer II: 250 mM NaCl
- Storage buffer I: 20 mM sodium acetate pH 6,5, 20% Ethanol
- Storage buffer II: 100 mM sodium hydrogen carbonate, 0.02% sodium azide, pH 7.5
- EDC: ((1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide) is hygroscopic and immediately starts hydrolyzing when getting in contact with humidity. It is highly recommended to open EDC bottles under protective gas and to let the EDC equilibrate to room temperature before opening. Use EDC directly after withdrawal from storage vessel! Important: If you are not sure if the EDC has been in contact with humidity, discard the material and use fresh EDC.
- 15 ml plastic tube.
- Centrifuge capable of 500xG for 15 ml tubes
- End-over-end shaker
Important: Never use buffers with free amines (e.g. tris) or carboxylate groups in EDC/NHS coupling reactions.
Depending on the nature of the protein, binding and wash buffers I or II might give best results.
|A.||Amine Coupling protocol|
- Dispense 2 ml of the 50% agarose suspension into a 15 ml plastic tube. Pellet the agarose by centrifugation at 500 x g, and remove the supernatant. Wash the agarose three times with 5 ml binding buffer I each.
- Resuspend 2-7 mg protein in 1 ml binding buffer I, add them to the agarose suspension and mix by vortexing. Incubate the reaction mixture for 5-10 minutes at 4°C on an end-over-end shaker.
- Equilibrate the EDC to room temperature (see above) and add 15 mg EDC directly to the suspension and mix immediately by vortexing. Incubate for 1h at room temperature – or for 2 h at 4°C if the protein is temperature-sensitive- on an end-over-end shaker.
- In some cases, the binding capacity can be raised by a second addition of 10 mg EDC and additional 1-2h incubation.
- Centrifuge the solution at 500 x g and discard the supernatant. Wash the agarose six times with 5 ml Wash Buffer I each. Add 1 ml storage buffer to obtain a 50% suspension for storage at 4°C and further use.
- Please contact us (firstname.lastname@example.org) if you have any questions or need assistance optimizing a protocol for your application.