Coupling Procedure for PureCube Amine Activated Agarose

 

Buffer compositions - Amine coupling protocol for biomolecules

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, coupling and wash buffers I or II might give best results

Coupling buffer I (PBS), pH 7.2

 
ComponentFinal concentration
Na phosphate 150 mM
NaCl 100 mM

Coupling buffer II, pH 4.7

 
ComponentFinal concentration
MES 0.1 M
NaCl 150 mM

Wash buffer I (PBS), pH 7.2

 
ComponentFinal concentration
Na phosphate 150 mM
NaCl 100 mM

Wash buffer II

 
ComponentFinal concentration
NaCl 250 mM

Storage buffer I, pH=6.5

 
ComponentFinal concentration
Sodium acetate 20 mM
Ethanol 20%

Storage buffer II, pH=7.5

 
ComponentFinal concentration
Sodium hydrogen carbonate 100 mM
Sodium azide 0.02%
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.
   
A.Amine Coupling protocol
 
 
  1. 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 coupling buffer I each.
  2. Resuspend 2-7 mg protein in 1 ml coupling 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.
  3. 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.
  4. In some cases, the binding capacity can be raised by a second addition of 10 mg EDC and additional 1-2h incubation.
  5. 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.
  6. Please contact us ([email protected]) if you have any questions or need assistance optimizing a protocol for your application.
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