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
| |
| Component | Final concentration |
|---|
| Na phosphate | 150 mM |
| NaCl | 100 mM |
Coupling buffer II, pH 4.7
| |
| Component | Final concentration |
|---|
| MES | 0.1 M |
| NaCl | 150 mM |
Wash buffer I (PBS), pH 7.2
| |
| Component | Final concentration |
|---|
| Na phosphate | 150 mM |
| NaCl | 100 mM |
Wash buffer II
| |
| Component | Final concentration |
|---|
| NaCl | 250 mM |
Storage buffer I, pH=6.5
| |
| Component | Final concentration |
|---|
| Sodium acetate | 20 mM |
| Ethanol | 20% |
Storage buffer II, pH=7.5
| |
| Component | Final concentration |
|---|
| Sodium hydrogen carbonate | 100 mM |
| Sodium azide | 0.02% |
| | |
| A. | Amine coupling protocol: |
|---|
| | - Dispense 800 µl of the 25% magbead suspension into a 2 ml microtube and place the tube in a MagBead seperator
- Remove the supernatant and wash the magbeads three times with 1 ml PBS each.
- Resuspend 400 µg to 2 mg protein in 200 µl PBS, add them to the suspension and mix by vortexing.
- Incubate the reaction mixture for 5-10 minutes at 4°C on an end-over-end shaker.
- Depending on the stability of your protein, incubate for 2h at 4°C or 1h at room temperature on an end-over-end shaker.
- In some cases, the binding capacity can be raised by a second addition of 4 mg EDC and additional 1-2h incubation.
- 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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