Coupling Procedure for PureCube Amine Activated MagBeads

 

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, binding 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%
   
A.Amine coupling protocol:
 
 
  1. Dispense 800 µl of the 25% magbead suspension into a 2 ml microtube and place the tube in a MagBead seperator
  2. Remove the supernatant and wash the magbeads three times with 1 ml PBS each.
  3. Resuspend 400 µg to 2 mg protein in 200 µl PBS, add them to the suspension and mix by vortexing.
  4. Incubate the reaction mixture for 5-10 minutes at 4°C on an end-over-end shaker.
  5. Depending on the stability of your protein, incubate for 2h at 4°C or 1h at room temperature on an end-over-end shaker.
  6. In some cases, the binding capacity can be raised by a second addition of 4 mg EDC and additional 1-2h incubation.
  7. Wash the mag beads five times with 1 ml PBS and once with 1 ml double distilled water. Resuspend the magbeads in 800 µl storage buffer. Keep at 4°C until further use.
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